Methods and compositions for treating kidney disorders

ABSTRACT

The present invention provides methods for treating glomerulosclerosis such as focal segmental glomerulosclerosis (FSGS) or glomerulonephritis such as immunoglobulin A nephropathy (IgAN) by cyclohexenone compounds.

CROSS-REFERENCE

This application claims the benefit of U.S. application Ser. No.61/435,201, filed Jan. 21, 2011, and U.S. application Ser. No.61/544,910, filed Oct. 7, 2011, each of which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Many diseases or disorders affect kidney function by attacking theglomeruli. Glomerular diseases include many conditions with a variety ofgenetic and environmental causes, but they fall into two majorcategories, Glomerulonephritis and Glomerulosclerosis.

Glomerulosclerosis refers to a hardening of the glomerulus in thekidney. It is a general term to describe scarring of the kidneys' tinyblood vessels, the glomeruli, the functional units in the kidney thatfilter urine from the blood. Proteinuria (large amounts of protein inurine) is one of the signs of glomerulosclerosis. Scarring disturbs thefiltering process of the kidneys and allows protein to leak from theblood into urine. However, glomerulosclerosis is one of many causes ofproteinuria. A kidney biopsy may be necessary to determine whether apatient has glomerulosclerosis or another kidney problem.Glomerulosclerosis, more specifically, can refer to focal segmentalglomerulosclerosis (FSGS) and nodular diabetic glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is defined by thecharacteristic lesions of focal glomerular sclerosis and foot processeffacement. The reported frequency of end-stage renal disease inpatients with FSGS ranges widely from 13 to 78% in studies with up to 20years of follow-up. Although the etiology and pathogenesis of FSGSremains unclear, it is believed to mainly arise from an intrinsic insultto the glomerular epithelial cell that activates complex interactionswithin the glomerulus, whereby resulting in glomerulosclerosis.

Nodular diabetic glomerulosclerosis or intercapillaryglomerulonephritis, also known as diabetic nephropathy (nephropatiadiabetica) or Kimmelstiel-Wilson syndrome is a progressive kidneydisease caused by angiopathy of capillaries in the kidney glomeruli. Itis characterized by nephrotic syndrome and diffuse glomerulosclerosis.It is due to longstanding diabetes mellitus, and is a prime indicationfor dialysis in many countries.

At present, although corticosteroids and immunemodulatory agents arecommonly used to treat patients with primary FSGS, the outcome oftherapy in terms of progression of the renal lesions is poor, inaddition to their various side effects and these regimens of treatmentare based more on empirical assumptions than pathogenetic evidence. (Seefor example, Matalon, et al., Semin Nephrol, 20: 309-317, 2000; Braun,et al., Cochrane Database Syst Rev: CD003233, 2008).

Glomerulonephritis describes the inflammation of the membrane tissue inthe kidney that serves as a filter, separating wastes and extra fluidfrom the blood.

Acceleration and progression during the course of Immunoglobulin Anephropathy (IgAN), the most common type of primary glomerulonephritis,is relatively unpredictable and clinically remains a challenge in termsof prophylaxis and treatment, and has been considered to be a key stepin subsequent development of chronic renal failure of the glomerulardisorder. In this regard, abnormal enhancement of both systemic T cellactivation and lymphocyte/macrophage/neutrophil infiltration in thekidney of IgAN patients has been considered as a major detrimentalprocess in converting IgAN into chronic renal failure (Kamei, et al.,Clin. J. Am. Soc. Nephrol. 2011, 14; Chan, et al., Clin. Exp. Nephrol.2004, 8:297-303; Chao, et al., Kidney Int. 70:283-297 (2006); Lai, K.N., Nephron. 92:263-270 (2002)), although other immunological, clinical,and pathological factors may also be attributable. Furthermore,oxidative stress has been highly implicated in the development andprogression of IgAN in patients and animal models; reactive oxygenspecies (ROS) has been reported to play an immediate pathogenic role inthe development of a wide range of human and experimental glomerulardisorders, including IgAN.

Although glucocorticoid steroids has been employed to treat IgANpatients, their efficacy of preserving renal function and reducingproteinuria in IgAN remains unclear, and adverse side effects are stilla problematic concern because of potential uncontrollableimmunosuppressive effects for long term use.

SUMMARY OF THE INVENTION

In one aspect provides herein methods for the treatment of glomerulardiseases (e.g., glomerulosclerosis or glomerulonephritis) in a subjectcomprising administering to the subject an effective amount of acyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In another aspect provides herein methods for attenuating renaldysfunction or glomerular lesions in a subject comprising administeringto the subject an effective amount of a cyclohexenone compound havingthe structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In another aspect provides herein methods for enhancing renal nuclearfactor E2-related factor 2 (Nrf2) activity in a subject comprisingadministering to the subject an effective amount of a cyclohexenonecompound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In another aspect provides herein methods for inhibiting renal NF-κBactivation and/or transforming growth factor (TGF)-β1 protein expressionin a subject comprising administering to the subject an effective amountof a cyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In another aspect provides herein methods for inhibiting ROS/NO and/orp47^(phox) in a subject comprising administering to the subject aneffective amount of a cyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In another aspect provides herein methods for reducing CD3⁺/CD69⁺ Tcells in a subject comprising administering to the subject an effectiveamount of a cyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In another aspect provides herein methods for enhancing glutathioneperoxidase (GPx) activity in the kidney comprising administering to asubject an effective amount of a cyclohexenone compound having thestructure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In another aspect provides herein methods for reducing pro-inflammatorycytokines in a subject comprising administering to the subject aneffective amount of a cyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In another aspect provides herein methods for reducing renal caspase-1protein expression and/or inhibiting renal NLRP3 activation in thekidney comprising administering to a subject an effective amount of acyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In yet another aspect provides herein methods for reducing renal NF-κBlevel in the kidney comprising administering to a subject an effectiveamount of a cyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In yet another aspect provides herein method for inhibiting apoptosis inthe kidney comprising administering to a subject an effective amount ofa cyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In another aspect provides herein methods for protecting or preventingkidney from glomerulosclerosis and/or interstitial fibrosis and/orglomerulonephritis in a subject comprising administering to the subjectan effective amount of a cyclohexenone compound having the structure

which decreases the expression levels of TGF-β1 protein and collagen I,III and IV protein accumulation in the kidney, wherein each of X and Yindependently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In yet another aspect provides methods for the treatment of focalsegmental glomerulosclerosis (FSGS) in a subject, which comprisesadministering to the subject a therapeutically effective amount of acyclohexenone compound having the structure

that (i) enhances Nrf2 activity and/or (ii) suppresses NF-κB-dependentinflammatory and TGF-β1-mediated fibrosis in the kidney, wherein

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In yet another aspect provides methods for the treatment ofglomerulonephritis in a subject, which comprises administering to thesubject a therapeutically effective amount of a cyclohexenone compoundhaving the structure

that (i) blocking renal NLRP3 inflammasome activation and/or (ii)inhibiting the increase in T cell activation, wherein each of X and Yindependently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In yet another aspect provides methods for maintaining immunoglobulin Anephropathy (IgAN) in remission in a subject, which comprisesadministering to the subject an effective amount of a cyclohexenonecompound having the structure

that (i) enhances Nrf2 activity and/or (ii) suppresses NF-κB-dependentinflammatory and TGF-β1-mediated fibrosis in the kidney,

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A-C show illustrative results from the exemplary cyclohexenoneCompound 1 to reduce urinary protein and improve renal function. (1A)Urinary protein time-course studies. (1B) Serum blood urea nitrogen(BUN) levels. (1C) Serum creatinine levels. The data are the mean±SEMfor six mice per group. *p<0.05, **p<0.01, ***p<0.005. #Not detectable.

FIGS. 2A-B show illustrative results of the renal histopathologydevelopment prevention by the exemplary cyclohexenone Compound 1. (2A)Kidney histopathological evaluation by H&E staining on day 7, 14, and 21of treatment. (2B) Detection of podocyte injury in glomeruli byimmunohistochemical staining of desmin on day 7, 14, and 21 oftreatment. The black arrow head, white arrow head, and arrow indicatethe epithelial hyperplasia lesions (EPHLs), sclerosis, and podocytes,respectively. Original magnification, 400×. Semi-quantitative analysisis shown in the right panel. The data are the mean±SEM for six mice pergroup. *p<0.05, **p<0.01, ***p<0.005. #Not detectable.

FIGS. 3A-F show illustrative results that an exemplary cyclohexenoneCompound 1 protects against ROS/NO production in FSGS mice. (3A)Superoxide anion levels in serum. (3B) NO levels in serum. (3C)Superoxide anion levels in urine. (3D) NO levels in urine. (3E)Superoxide anion levels in kidney protein. (3F) kidney in-situ ROSproduction demonstrated by dihydroethidium (DHE) labeling. Originalmagnification, 400×. Semi-quantitative analysis is shown in the rightpanel. The data are the mean±SEM for six mice per group. *p<0.05,**p<0.01, ***p<0.005.

FIGS. 4A-E show illustrative results from the exemplary cyclohexenoneCompound 1 enhancing nuclear Nrf2 expression and decreasing cytosolicp47^(phox) expression in the kidney. (4A) Representative Western blotsof cytosolic p47^(phox) and (4B) nuclear Nrf2 in kidney tissues. β-Actinand Histone H3 were used as internal controls for cytosolic and nuclearproteins, respectively. (4C) Quantification of the p47^(phox)/β-actinratio and (4D) the Nrf2/Histone H3 ratio. (4E) GPx activity in thekidney. The data are the mean±SEM for six mice per group. *p<0.05,**p<0.01, ***p<0.005.

FIGS. 5A-B show illustrative results of T cell and macrophageinfiltration with the exemplary cyclohexenone Compound 1. (5A) Detectionof CD3⁺ T cells or (5B) F4/80 monocytes/macrophages byimmunohistochemical staining Original magnification, 400×. The red arrowindicate the CD3⁻ T cells. Semi-quantitative analysis is shown in theright panel. The data are the mean±SEM for six mice per group. *p<0.05,**p<0.01, ***p<0.005.

FIGS. 6A-C show illustrative results from the exemplary cyclohexenoneCompound 1 suppressing IL-6 expression and NF-κB activation in thekidney. (6A) Detection of IL-6 protein and (6B) NF-κB p65 byimmunohistochemical staining Original magnification, 400×.Semi-quantitative analysis is shown in the lower panel. (4C) KidneyNF-κB activity. The data are the mean±SEM for six mice per group.*p<0.05, **p<0.01, ***p<0.005.

FIGS. 7A-C show illustrative results from the exemplary cyclohexenoneCompound 1 for the prevention of collagen I, III and IV accumulation inthe kidney. (7A) Detection of collagen I, (7B) collagen III, or (7C)collagen IV by immunohistochemical staining Original magnification,400×. Semi-quantitative analysis is shown in the right panel. The dataare the mean±SEM for six mice per group. **p<0.01, ***p<0.005.

FIGS. 8A-C show illustrative results from the exemplary cyclohexenoneCompound 1 for the prevention of TGF-β1 expression in serum and kidneytissues. (8A) TGF-β1 levels in serum. (8B) TGF-β1 levels in kidneyprotein. (8C) Detection of TGF-β1 by immunohistochemical stainingOriginal magnification, 400×. Semi-quantitative analysis is shown in theright panel. The data are the mean±SEM for six mice per group. *p<0.05,**p<0.01, ***p<0.005.

FIGS. 9A-9E show illustrative results from the exemplary cyclohexenoneCompound 1 to reduce urinary protein and improve renal function andsevere renal histopathology in AcP-IgAN mice. (9A) Urine proteintime-course studies. (9B) Serum blood urea nitrogen (BUN) levels. (9C)Serum creatinine levels. Kidney histopathological evaluation by H&Estaining (9D) and PAS staining (9E) at day 3 and 28 of treatment.Original magnification, 400×. The scoring of the percentage of glomeruliaffected by the indicated parameter is shown in the lower panels. Thedata are the mean±SEM for six mice per group. *p<0.05, **p<0.01,***p<0.005. #Not detectable.

FIGS. 10A-D show illustrative results of both mRNA and protein levels ofTGF-β1 and Col-IV in the AcP-IgAN mice fed with the exemplaryCompound 1. Detection renal mRNA levels of TGF-β1 (10A) and collagen I(10B) by real-time PCR. Detection renal protein levels of TGF-β1 (10C)and collagen I (10D) by immunohistochemical staining. Originalmagnification, 400×. The scoring is shown in the lower panels. The dataare the mean±SEM for six mice per group. **p<0.01, ***p<0.005.

FIGS. 11A-C show illustrative results of cell mediated immunity in thepathogenesis of IgAN by cytometry in splenocytes. Percentage ofCD3⁺CD69⁺ cells in CD3⁺ splenocytes (11A) or CD19⁻CD69⁺ cells in CD19⁺splenocytes (11B) at day 3 and 28 of treatment. (11C) T cellproliferation. The data are the mean±SEM for six mice per group.*p<0.05, **p<0.01, ***p<0.005. #Not detectable.

FIGS. 12A-F show illustrative results of evaluation of the phenotypicexpression of mononuclear leukocytes that infiltrated in the kidney ofthe Acp-IgAN mice. (12A-C) Detection of CD3⁺ T cells (12A), CD4⁺ T cells(12B), or CD8⁺ T cells (12C) by immunofluorescence staining (12D-F)Detection of CD11b macrophages/neutrophils (12D), CD11c dendritic cells(12E), or F4/80 monocytes/macrophages (12F) by immunohistochemicalstaining Original magnification, 400×. The scoring is shown in the lowerpanels. The data are the mean±SEM for six mice per group. *p<0.05,**p<0.01, ***p<0.005. #Not detectable.

FIGS. 13A-F show illustrative results that the exemplary cyclohexenoneCompound 1 protects against ROS/NO production in AcP-IgAN mice. (FIGS.13A and 13B) Serum levels of superoxide anion (13A) or NO (13B). (FIGS.13C and 13D) Urine levels of superoxide anion (13C) or NO (13D). (FIG.13E) Superoxide anion levels in the kidney. (FIG. 13F) Kidney in situROS production demonstrated by dihydroethidium (DHE) labeling. Originalmagnification, 400×. The scoring is shown in the right panel. The dataare the mean±SEM for six mice per group. *p<0.05, **p<0.01, ***p<0.005.

FIGS. 14A-F show illustrative results of the expression levels of bothmRNA and protein of Nrf2 in AcP-IgAN mice. (14A-C) Detection renal mRNAlevels of Nrf2 (14A), NQO1 (14B), or HO-1 (14C) by real-time PCR.(14D-E) Detection renal levels of nuclear Nrf2 (14D) or cytosolic HO-1(14E) by ELISA. (14F) GPx activity in the kidney. The data are themean±SEM for six mice per group. *p<0.05, **p<0.01, ***p<0.005.

FIGS. 15A-D show illustrative results of serum levels of inflammatorycytokines in AcP-IgAN mice. (15A) IL-6. (15B) MCP-1. (15C) IL-1β. (15D)IL-18. The data are the mean±SEM for six mice per group. *p<0.05,**p<0.01, ***p<0.005.

FIGS. 16A-F show illustrative results of renal NLRP3 inflammasomeactivation in AcP-IgAN mice. (16A-D) Detection renal mRNA levels ofNLRP3 (16A), caspase-1 (16B), IL-1β (16C), or IL-18 (16D) by real-timePCR. (16E-F) Representative Western blots of NLRP3 (16E) or caspase-1(Casp1) (16F) in kidney tissues. The appearance of the Casp1 p20 subunitindicates activation. β-actin was used as internal control. *p<0.05,**p<0.01, ***p<0.005.

FIGS. 17A-F show illustrative results from the exemplary cyclohexenoneCompound 1 suppressing IL-6 and MCP-1 expression and NF-κB activation inthe kidney of AcP-IgAN mice. (17A) Detection of NF-κB p65 byimmunohistochemical staining. Original magnification, 400×. The scoringis shown in the lower panel. (17B) Measurement of kidney NF-κB activityusing an ELISA-based TransAM NF-κB kit. (17C-D) Detection renal mRNAlevels of MCP-1 (17C) and IL-6 (17D) by real-time PCR. (17E-F) Detectionrenal protein levels of MCP-1 (17E) and IL-6 (17F) byimmunohistochemical staining. Original magnification, 400×. The scoringis shown in the lower panels. The data are the mean±SEM for six mice pergroup. *p<0.05, **p<0.01, ***p<0.005.

FIGS. 18A-B show illustrative results of apoptosis in the kidney ofAcP-IgAN mice. (A) Apoptosis was detected in the kidney by terminaldeoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL).Original magnification, 400×. (B) Scoring of apoptosis-positive cells inthe kidney. The data are the mean±SEM for six mice per group. *p<0.05,**p<0.01, ***p<0.005.

DETAILED DESCRIPTION OF THE INVENTION

Glomerular diseases include many conditions with a variety of geneticand environmental causes, but they fall into two major categories,Glomerulonephritis and Glomerulosclerosis. Glomerulosclerosis especiallyFSGS is believed to mainly arise from an intrinsic insult to theglomerular epithelial cell that activates complex interactions withinthe glomerulus. These complex interactions may include oxidative stress,inflammation with macrophage recruitment, and factors promoting matrixproduction and/or matrix degradation. High levels of both systemic Tcell activation and neutrophil/lymphocyte/macrophage infiltration in thekidney has been increasingly implicated in the acceleration andprogression of immunoglobulin A nephropathy (IgAN), the most frequenttype of primary glomerulonephritis. So far, however, both prevention andtreatment for an aggressive and exacerbated stage of IgAN remainslargely under investigation. Provided herein are methods for thetreatment of kidney disorders, especially glomerulosclerosis orglomerulonephritis by administering a cyclohexenone compound providedherein to a subject (e.g. a human). The cyclohexenone compound providestherapeutic effects to a subject (especially in the kidney) for treatingglomerulosclerosis (see Examples 1-6, and 14) and/or glomerulonephritis(see Examples 7-13, and 14).

In some embodiments, there are provided methods for the treatment ofglomerular diseases (such as glomerulosclerosis or glomerulonephritis)in a subject. The methods comprise administering to the subject atherapeutically effective amount of a cyclohexenone compound having thestructure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In some embodiments, there are provided methods for the treatment ofglomerulosclerosis. In certain embodiments, glomerulosclerosis is focalsegmental glomerulosclerosis (FSGS) or nodular diabeticglomerulosclerosis. In certain embodiments, glomerulosclerosis is focalsegmental glomerulosclerosis (FSGS). In some embodiments, thecyclohexenone compound blocks oxidative stress. In certain embodiments,the oxidative stress is blocked by reducing TGF-β1 and extracellularmatrix protein expression. In some embodiments, the subject is human. Incertain embodiments, the oxidative stress is reduced by enhancingnuclear factor E2-related factor 2 (Nrf2) activity.

In some embodiments, there are provided methods for the treatment ofglomerulonephritis. In certain embodiments, glomerulonephritis isimmunoglobulin A nephropathy (IgAN). In some embodiments, thecyclohexenone compound reduces CD3⁺/CD69⁺ T cells in the subject. Incertain embodiments, the cyclohexenone compound reduces pro-inflammatorycytokines in the subject. In certain embodiments, the pro-inflammatorycytokines comprise MCP-1, IL-6, IL-1β, IL-18, or combinations thereof.In some embodiments, the subject is human.

In some embodiments, the cyclohexenone compound having the structure

is prepared synthetically or semi-synthetically from any suitablestarting material. In other embodiments, the cyclohexenone compound isprepared by fermentation, or the like. For example, Compound 1 (alsoknown as Antroquinonol® or “Antroq”) or Compound 3, in some instances,is prepared from 4-hydroxy-2,3-dimethoxy-6-methylcyclohexa-2,5-dienone.The non-limited exemplary compounds are illustrated below.

In other embodiments, the cyclohexenone compound having the structure

is isolated from the organic solvent extracts of Antrodia camphorate. Insome embodiments, the organic solvent is selected from alcohols (e.g.,methanol, ethanol, propanol, or the like), esters (e.g., methyl acetate,ethyl acetate, or the like), alkanes (e.g., pentane, hexane, heptane, orthe like), halogenated alkanes (e.g., chloromethane, chloroethane,chloroform, methylene chloride, and the like), and the like. Forexample, exemplary Compounds 1-7 are isolated from organic solventextracts. In certain embodiments, the organic solvent is alcohol. Incertain embodiments, the alcohol is ethanol. In some embodiments, thecyclohexenone compound is isolated from the aqueous extracts of ofAntrodia camphorate.

In some embodiments, there are provided methods for attenuating renaldysfunction or glomerular lesions in a subject comprising administeringto the subject an effective amount of a cyclohexenone compound havingthe structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.        In some embodiments, the glomerular lesions comprise epithelial        hyperplasia lesion (EPHL). In some embodiments, the subject is        human.

In some embodiments, the cyclohexenone compounds provided herein possessthe therapeutic effects of enhancing nuclear factor E2-related factor 2(Nrf2) activity but suppressing NF-κB-dependent inflammatory andTGF-β1-mediated fibrosis pathways in the kidney. See Examples 5, 6 and14.

In some embodiments provide methods for enhancing renal nuclear factorE2-related factor 2 (Nrf2) activity in a subject comprisingadministering to the subject an effective amount of a cyclohexenonecompound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.        In some embodiments, the subject is human.

In some embodiments provide methods for inhibiting renal NF-κBactivation and/or transforming growth factor (TGF)-β1 protein expressionin a subject comprising administering to the subject an effective amountof a cyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.        In some embodiments, the subject is human.

In some embodiments, administration of the cyclohexenone compoundprovided herein (e.g., Compound 1) inhibits ROS/NO and p47^(phox)NAD(P)H oxidase production in the kidney, but clearly enhances Nrf2signaling pathway responsible for the effects of the cyclohexenonecompound on the FSGS subject. See Examples 3, 5 and 14.

In some embodiments provide methods for inhibiting ROS/NO and/orp47^(phox) in a subject comprising administering to the subject aneffective amount of a cyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.        In some embodiments, the subject is human.

In some embodiments provide methods for reducing CD3⁺/CD69⁺ T cells in asubject comprising administering to the subject an effective amount of acyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In some embodiments, the subject is human.

It was observed that the control FSGS mice showed significantly enhancedGPx activity on day 7 after the disease induction (Examples 4 and 14).Oxidative stress and its constant companion inflammation are also commonfeatures of chronic kidney disease (Kim, et al., Am J Physiol RenalPhysiol, 298: F662-671, 2010; Yoon, et al., Kidney Int, 71: 167-172,2007) and play a critical part in progression of glomerularsclerosis. Ofnote, oxidative stress and inflammation are intimately related as eachof them recruits and amplifies the other to trigger a vicious cycle. Forinstance, oxidative stress can induce inflammation by activating NF-κBand subsequent production of pro-inflammatory cytokines and chemokineswhereby leading to leukocyte activation and production and releasingROS/NO, while these events promote oxidative stress in return.(Anrather, et al., J Biol Chem, 281: 5657-5667, 2006; Vaziri, et al.,Nat Clin Pract Nephrol, 2: 582-593, 2006; Rodrigo, et al., Free RadicBiol Med, 33: 409-422, 2002) In addition, the inflammatory response toactivation of NF-κB and consequent induction of cyclooxygenase-2,inducible nitric oxide synthase, IL-6, and TNF-α is more intense in Nrf2knockout mice compared with the wild type mice. (Chen, et al., Am JPhysiol Heart Circ Physiol, 290: H1862-1870, 2006; Li, et al., BiochemPharmacol, 76: 1485-1489, 2008) It also has been reported thatdeficiency of HO-1, which is regulated by Nrf2, has been shown toaccentuate glomerulonephritis. (Datta, et al., J Am Soc Nephrol, 10:2540-2550, 1999)

In some embodiments, administration of the cyclohexenone compoundprovided herein (e.g., Compound 1) significantly reduces renal IL-6expression and blocks NF-κB activation in the kidney. This was confirmedby significantly inhibited infiltration of T cells and macrophages intothe kidney in the FSGS+Antroq mice. (Examples 5, 11 and 14) In someembodiments, the effect is a mechanism responsible for preventinginterstitial inflammation and EPHLs, the latter being a key index forthe renal progression of FSGS. (D'Agati, V Semin Nephrol, 23: 117-134,2003; Nagata, et al., Lab Invest, 80: 869-880, 2000)

In some embodiments provide methods for enhancing glutathione peroxidase(GPx) activity in the kidney comprising administering to a subject aneffective amount of a cyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.        In some embodiments, the subject is human.

In some embodiments provide methods for reducing pro-inflammatorycytokines in a subject comprising administering to the subject aneffective amount of a cyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.        In certain embodiments, the pro-inflammatory cytokines comprise        MCP-1, IL-6, IL-1β, IL-18, or combinations thereof. In some        embodiments, the subject is human.

In some embodiments provide methods for reducing renal caspase-1 proteinexpression and/or inhibiting renal NLRP3 activation in the kidneycomprising administering to a subject an effective amount of acyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.        The subject, in some embodiments, is human.

In some embodiments provide methods for reducing renal NF-κB activationin the kidney comprising administering to a subject an effective amountof a cyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.        In some embodiments, the subject is human.

In some embodiments, there are provided methods for inhibiting apoptosisin the kidney comprising administering to a subject an effective amountof a cyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.        The subject in some instances is human.

In some embodiments, there are provided methods for protecting orpreventing kidney from glomerulosclerosis and/or interstitial fibrosisand/or glomerulonephritis in a subject comprising administering to thesubject an effective amount of a cyclohexenone compound having thestructure

which decreases the expression levels of TGF-β1 protein and collagen I,III and IV protein accumulations in the kidney,

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.        In some embodiments, the subject is human.

Oxidative stress caused by combination of increased production ofreactive oxygen species (ROS) and/or nitric oxide (NO) and impairedantioxidant capacity, leads to promote necrosis, apoptosis,inflammation, fibrosis, and other disorders of kidney. (See for example,Kim, et al., Am J Physiol Renal Physiol, 298: F662-671, 2010) Recentadvance in the dissection of renal fibrosis/sclerosis mechanisms hasprovided evidence that NAD(P)H oxidase enzyme complex of infiltratingleukocytes and intrinsic renal cells plays an important role in theproduction of superoxide in renal lesions. (IBID; Jones, et al., J AmSoc Nephrol, 5: 1483-1491, 1995; Radeke, et al., J Biol Chem, 266:21025-21029, 1991) Blockade of oxidative stress can ameliorate renalsclerosis through anti-inflammatory and anti-apoptosis process. Besides,nuclear factor E2-related factor 2 (Nrf2) is found to be a criticaltranscription factor that binds to the antioxidant response element inthe promoter region of a number of genes encoding numerous antioxidantand phase 2 enzymes, such as glutathione peroxidase (GPx), catalase, andsuperoxide dismutase, in several types of cells and tissues. (Itoh, etal., Biochem Biophys Res Commun, 236: 313-322, 1997; Nguyen, et al., JBiol Chem, 284: 13291-13295, 2009) The Nrf2-mediated regulation ofcellular antioxidant production and anti-inflammatory machinery play animportant role against oxidative stress, and Nrf2 signaling pathway hasbeen demonstrated to play a protective role against renal fibrosis inrat tubular epithelial cells (Shin, et al., Free Radic Biol Med, 48:1051-1063, 2010) and streptozotocin-induced diabetic nephropathy (Jiang,et al., Diabetes, 59: 850-860, 2010) via transforming growth factor(TGF)-β1 associated epithelial-mesenchymal transition.

The expression of profibrotic cytokines, particularly TGF-β1 is pivotaldeterminants for renal sclerosis/fibrosis. (Ka, et al., J Am SocNephrol, 18: 1777-1788, 2007; Lan, H Y. Front Biosci, 13: 4984-4992,2008; Zhao, et al., Am J Nephrol, 28: 548-554, 2008) Blocking ofoxidative stress has been reported to ameliorate glomerulosclerosis byreducing TGF-β1 and extracellular matrix proteins expression.(Hahn, etal., Pediatr Nephrol, 13: 195-198, 1999; Kashihara, et al., Curr MedChem.; Manning, et al., Am J Nephrol, 25: 311-317, 2005) Also,anti-oxidative stress signaling pathway involving Nrf2 transcriptionfactor and its related phase II enzymes has been shown to play a renalprotective role against fibrosis in rat tubular epithelial andstreptozotocin-induced diabetic nephropathy.

In some embodiments, treatment with the cyclohexenone compound providedherein (e.g., Compound 1) decreases the expression levels of TGF-β1protein and its downstream collagen I, III, and IV protein accumulationsin the kidney (Examples 6 and 14); this suggests the cyclohexenonecompound provided herein is able to protect kidney fromglomerulosclerosis and interstitial fibrosis as shown in the treatedFSGS mice by blocking TGF-β1-mediated fibrosis pathway.

In some embodiments provide methods for the treatment of focal segmentalglomerulosclerosis (FSGS), which comprises administering to a subject aneffective amount of a cyclohexenone compound having the structure

that (i) enhances Nrf2 activity and/or (ii) suppresses NF-κB-dependentinflammatory and TGF-β1-mediated fibrosis in the kidney,

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.        In some embodiments, the subject is human.

In some embodiments provide methods for the treatment ofglomerulonephritis in a subject, which comprises administering to thesubject an effective amount of a cyclohexenone compound having thestructure

that (i) blocking renal NLRP3 inflammasome activation and/or (ii)inhibiting the increase in T cell activation, wherein each of X and Yindependently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

In accordance with the present invention, the exemplary cyclohexenoneCompound 1 altered T cell activity and prevented renal inflammation inthe AcP-IgAN mice, the exemplary cyclohexenone compounds are suitable tomaintain IgAN in remission.

In certain embodiments, provided herein are methods for maintainingimmunoglobulin A nephropathy (IgAN) in remission in a subject, whichcomprises administering to the subject an effective amount of acyclohexenone compound having the structure

wherein each of X and Y independently is oxygen, NR₅ or sulfur;

-   -   R is a hydrogen or C(═O)C₁-C₈alkyl;    -   each of R₁, R₂ and R₃ independently is a hydrogen, methyl or        (CH₂)_(m)—CH₃;    -   R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, halogen, 5 or        6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl,        aryl, glucosyl, wherein the 5 or 6-membered lactone, C₁-C₈alkyl,        C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl are optionally        substituted with one or more substituents selected from NR₅R₆,        OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,        C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈        alkoxy;    -   each of R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl;    -   R₇ is a C₁-C₈alkyl, OR₅ or NR₅R₆;    -   m=1-12; and    -   n=1-12; or a pharmaceutically acceptable salt, metabolite,        solvate or prodrug thereof.

Certain Terminology

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Unlessotherwise indicated, conventional methods of mass spectroscopy, NMR,HPLC, protein chemistry, biochemistry, recombinant DNA techniques andpharmacology are employed. In this application, the use of “or” or “and”means “and/or” unless stated otherwise. Furthermore, use of the term“including” as well as other forms, such as “include”, “includes,” and“included,” is not limiting. The section headings used herein are fororganizational purposes only and are not to be construed as limiting thesubject matter described.

An “alkyl” group refers to an aliphatic hydrocarbon group. The alkylgroup may be a saturated alkyl group (which means that it does notcontain any carbon-carbon double bonds or carbon-carbon triple bonds) orthe the alkyl group may be an unsaturated alkyl group (which means thatit contains at least one carbon-carbon double bonds or carbon-carbontriple bond). The alkyl moiety, whether saturated or unsaturated, may bebranched, or straight chain.

The “alkyl” group may have 1 to 10 carbon atoms (whenever it appearsherein, a numerical range such as “1 to 10” refers to each integer inthe given range; e.g., “1 to 10 carbon atoms” means that the alkyl groupmay consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., upto and including 10 carbon atoms, although the present definition alsocovers the occurrence of the term “alkyl” where no numerical range isdesignated). The alkyl group of the compounds described herein may bedesignated as “C₁-C₆ alkyl” or similar designations. By way of exampleonly, “C₁-C₆ alkyl” indicates that there are one, two, three, four,five, or six carbon atoms in the alkyl chain. In one aspect the alkyl isselected from the group consisting of methyl, ethyl, propyl, iso-propyl,n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groupsinclude, but are in no way limited to, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tertiary butyl, pentyl, neopentyl, hexyl,allyl, but-2-enyl, but-3-enyl, cyclopropylmethyl, cyclobutylmethyl,cyclopentylmethyl, cyclohexylmethyl, and the like. In one aspect, analkyl is a C₁-C₆ alkyl.

The term “alkylene” refers to a divalent alkyl radical. Any of the abovementioned monovalent alkyl groups may be an alkylene by abstraction of asecond hydrogen atom from the alkyl. In one aspect, an alkylene is aC₁-C₆alkylene. In another aspect, an alkylene is a C₁-C₄alkylene.Typical alkylene groups include, but are not limited to, —CH₂—,—CH(CH₃)—, —C(CH₃)₂—, —CH₂CH₂—, —CH₂CH(CH₃)—, —CH₂C(CH₃)₂—, —CH₂CH₂CH₂—,—CH₂CH₂CH₂CH₂—, and the like.

As used herein, the term “aryl” refers to an aromatic ring wherein eachof the atoms forming the ring is a carbon atom. Aryl rings are formed byfive, six, seven, eight, nine, or more than nine carbon atoms. Arylgroups are optionally substituted. In one aspect, an aryl is a phenyl ora naphthalenyl. In one aspect, an aryl is a phenyl. In one aspect, anaryl is a C₆-C₁₀aryl. Depending on the structure, an aryl group can be amonoradical or a diradical (i.e., an arylene group). In one aspect, anarylene is a C₆-C₁₀ arylene. Examplary arylenes include, but are notlimited to, phenyl-1,2-ene, phenyl-1,3-ene, and phenyl-1,4-ene.

The term “aromatic” refers to a planar ring having a delocalizedπ-electron system containing 4n+2π electrons, where n is an integer.Aromatic rings can be formed from five, six, seven, eight, nine, ten, ormore than ten atoms. Aromatics are optionally substituted. The term“aromatic” includes both carbocyclic aryl (“aryl”, e.g., phenyl) andheterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups (e.g.,pyridine). The term includes monocyclic or fused-ring polycyclic (i.e.,rings which share adjacent pairs of carbon atoms) groups.

The term “halo” or, alternatively, “halogen” or “halide” means fluoro,chloro, bromo or iodo.

The term “lactone” refers to a cyclic ester which can be seen as thecondensation product of an alcohol group —OH and a carboxylic acid group—COOH in the same molecule. It is characterized by a closed ringconsisting of two or more carbon atoms and a single oxygen atom, with aketone group ═O in one of the carbons adjacent to the other oxygen.

The term “heterocycle” or “heterocyclic” refers to heteroaromatic rings(also known as heteroaryls) and heterocycloalkyl rings (also known asheteroalicyclic groups) containing one to four heteroatoms in thering(s), where each heteroatom in the ring(s) is selected from O, S andN, wherein each heterocyclic group has from 4 to 10 atoms in its ringsystem, and with the proviso that the any ring does not contain twoadjacent O or S atoms. Non-aromatic heterocyclic groups (also known asheterocycloalkyls) include groups having only 3 atoms in their ringsystem, but aromatic heterocyclic groups must have at least 5 atoms intheir ring system. The heterocyclic groups include benzo-fused ringsystems. An example of a 3-membered heterocyclic group is aziridinyl. Anexample of a 4-membered heterocyclic group is azetidinyl. An example ofa 5-membered heterocyclic group is thiazolyl. An example of a 6-memberedheterocyclic group is pyridyl, and an example of a 10-memberedheterocyclic group is quinolinyl. Examples of non-aromatic heterocyclicgroups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, oxazolidinonyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl,thioxanyl, piperazinyl, aziridinyl, azetidinyl, oxetanyl, thietanyl,homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 1,2,3,6-tetrahydropyridinyl, pyrrolin-2-yl, pyrrolin-3-yl,indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl andquinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl,imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl,furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl,quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl,cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl,triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl,furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl,benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, andfuropyridinyl. The foregoing groups may be C-attached or N-attachedwhere such is possible. For instance, a group derived from pyrrole maybe pyrrol-1-yl (N-attached) or pyrrol-3-yl (C-attached). Further, agroup derived from imidazole may be imidazol-1-yl or imidazol-3-yl (bothN-attached) or imidazol-2-yl, imidazol-4-yl or imidazol-5-yl (allC-attached). The heterocyclic groups include benzo-fused ring systems.Non-aromatic heterocycles may be substituted with one or two oxo (═O)moieties, such as pyrrolidin-2-one.

Combination Treatments

In general, the compositions described herein and, in embodiments wherecombinational therapy is employed, other agents do not have to beadministered in the same pharmaceutical composition, and in someembodiments, because of different physical and chemical characteristics,are administered by different routes. In some embodiments, the initialadministration is made according to established protocols, and then,based upon the observed effects, the dosage, modes of administration andtimes of administration is modified by the skilled clinician.

In some embodiments, therapeutically-effective dosages vary when thedrugs are used in treatment combinations. Combination treatment furtherincludes periodic treatments that start and stop at various times toassist with the clinical management of the patient. For combinationtherapies described herein, dosages of the co-administered compoundsvary depending on the type of co-drug employed, on the specific drugemployed, on the disease, disorder, or condition being treated and soforth.

It is understood that in some embodiments, the dosage regimen to treat,prevent, or ameliorate the condition(s) for which relief is sought, ismodified in accordance with a variety of factors. These factors includethe disorder from which the subject suffers, as well as the age, weight,sex, diet, and medical condition of the subject. Thus, in otherembodiments, the dosage regimen actually employed varies widely andtherefore deviates from the dosage regimens set forth herein.

Combinations of compounds (i.e., the cyclohexenone compound describedherein) with other sterols and/or immunosuppressives are intended to becovered. In some embodiments, examples of immunosuppressives include,but are not limited to, the following: glucocorticoids, cytostatics,antibodies, drugs acting on immunophilins, and other drugs such asinterferons, opioids, TNF binding proteins and mycophenolate.

In some embodiments provide a composition for early treatment of kidneydisorders (such as glomerulonephritis, glomerulosclerosis, and the like)comprising an effective amount of a cyclohexenone compound having thestructure:

and one or more sterols and/or immunosuppressives, wherein each of X andY independently is oxygen, NR₅ or sulfur; R is a hydrogen orC(═O)C₁-C₈alkyl; each of R₁, R₂ and R₃ independently is a hydrogen,methyl or (CH₂)_(m)—CH₃, R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅,halogen, 5 or 6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl,C₂-C₈alkynyl, aryl, glucosyl, wherein the 5 or 6-membered lactone,C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl areoptionally substituted with one or more substituents selected fromNR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈ alkoxy; eachof R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl; R₇ is aC₁-C₈alkyl, OR₅ or NR₅R₆; m=1-12; and n=1-12; or a pharmaceuticallyacceptable salt, metabolite, solvate or prodrug thereof. In someembodiments, the kidney disorder is glomerulosclerosis (e.g., renalfibrosis or sclerosis in FSGS). In other embodiments, the kidneydisorder is glomerulonephritis (e.g., immunoglobulin A nephropathy(IgAN)).

“Glucocorticoids ” refers to a class of steroid hormones that bind tothe glucocorticoid receptor (GR), which is present in almost everyvertebrate animal cell. The name glucocorticoid derives from their rolein the regulation of the metabolism of glucose, their synthesis in theadrenal cortex, and their steroidal structure. Examples ofglucocorticoids include, but are not limited to, hydrocortisone(Cortisol), cortisone acetate, prednisone, prednisolone,methylprednisolone, dexamethasone, betamethasone, triamcinolone,beclometasone, fludrocortisone acetate, deoxycorticosterone acetate(DOCA), and aldosterone.

Examples of drugs acting on immunophilins include, but not limited tocyclosporin, tacrolimus, voclosporin and other calcineurin inhibitors,and sirolimus.

Certain Pharmaceutical and Medical Terminology

The term “acceptable” with respect to a formulation, composition oringredient, as used herein, means having no persistent detrimentaleffect on the general health of the subject being treated.

Antrodia is a genus of fungi in the family Meripilaceae. Antrodiaspecies have fruiting bodies that typically lie flat or spread out onthe growing surface, with the hymenium exposed to the outside; the edgesmay be turned so as to form narrow brackets. Most species are found intemperate and boreal forests, and cause brown rot. Some of the speciesin this genus are have medicinal properties, and have been used inTaiwan as a Traditional medicine.

The term “carrier,” as used herein, refers to relatively nontoxicchemical compounds or agents that facilitate the incorporation of acompound into cells or tissues.

The terms “co-administration” or the like, as used herein, are meant toencompass administration of the selected therapeutic agents to a singlepatient, and are intended to include treatment regimens in which theagents are administered by the same or different route of administrationor at the same or different time.

The term “diluent” refers to chemical compounds that are used to dilutethe compound of interest prior to delivery. Diluents can also be used tostabilize compounds because they can provide a more stable environment.Salts dissolved in buffered solutions (which also can provide pH controlor maintenance) are utilized as diluents in the art, including, but notlimited to a phosphate buffered saline solution.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an agent or a compoundbeing administered which will relieve to some extent one or more of thesymptoms of the disease or condition being treated. The result can bereduction and/or alleviation of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Forexample, an “effective amount” for therapeutic uses is the amount of thecomposition comprising a compound as disclosed herein required toprovide a clinically significant decrease in disease symptoms. Anappropriate “effective” amount in any individual case may be determinedusing techniques, such as a dose escalation study.

The terms “enhance” or “enhancing,” as used herein, means to increase orprolong either in potency or duration a desired effect. Thus, in regardto enhancing the effect of therapeutic agents, the term “enhancing”refers to the ability to increase or prolong, either in potency orduration, the effect of other therapeutic agents on a system. An“enhancing-effective amount,” as used herein, refers to an amountadequate to enhance the effect of another therapeutic agent in a desiredsystem.

A “metabolite” of a compound disclosed herein is a derivative of thatcompound that is formed when the compound is metabolized. The term“active metabolite” refers to a biologically active derivative of acompound that is formed when the compound is metabolized. The term“metabolized,” as used herein, refers to the sum of the processes(including, but not limited to, hydrolysis reactions and reactionscatalyzed by enzymes) by which a particular substance is changed by anorganism. Thus, enzymes may produce specific structural alterations to acompound. For example, cytochrome P450 catalyzes a variety of oxidativeand reductive reactions while uridine diphosphate glucuronyltransferasescatalyze the transfer of an activated glucuronic-acid molecule toaromatic alcohols, aliphatic alcohols, carboxylic acids, amines and freesulphydryl groups. Metabolites of the compounds disclosed herein areoptionally identified either by administration of compounds to a hostand analysis of tissue samples from the host, or by incubation ofcompounds with hepatic cells in vitro and analysis of the resultingcompounds.

The term “pharmaceutical combination” as used herein, means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. a compound (i.e., a cyclohexenone compound describedherein) and a co-agent, are both administered to a patientsimultaneously in the form of a single entity or dosage. The term“non-fixed combination” means that the active ingredients, e.g. acompound (i.e., a cyclohexenone compound described herein) and aco-agent, are administered to a patient as separate entities eithersimultaneously, concurrently or sequentially with no specificintervening time limits, wherein such administration provides effectivelevels of the two compounds in the body of the patient. The latter alsoapplies to cocktail therapy, e.g. the administration of three or moreactive ingredients.

The term “pharmaceutical composition” refers to a mixture of a compound(i.e., a cyclohexenone compound described herein) with other chemicalcomponents, such as carriers, stabilizers, diluents, dispersing agents,suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the compound toan organism. Multiple techniques of administering a compound exist inthe art including, but not limited to: intravenous, oral, aerosol,parenteral, ophthalmic, pulmonary and topical administration.

The term “subject” or “patient” encompasses mammals. Examples of mammalsinclude, but are not limited to, any member of the Mammalian class:humans, non-human primates such as chimpanzees, and other apes andmonkey species; farm animals such as cattle, horses, sheep, goats,swine; domestic animals such as rabbits, dogs, and cats; laboratoryanimals including rodents, such as rats, mice and guinea pigs, and thelike. In one embodiment, the mammal is a human.

The terms “treat,” “treating” or “treatment,” as used herein, includealleviating, abating or ameliorating at least one symptom of a diseaseor condition, preventing additional symptoms, inhibiting the disease orcondition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition eitherprophylactically and/or therapeutically.

Routes of Administration

Suitable routes of administration include, but are not limited to, oral,intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary,transmucosal, transdermal, vaginal, otic, nasal, and topicaladministration. In addition, by way of example only, parenteral deliveryincludes intramuscular, subcutaneous, intravenous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intraperitoneal, intralymphatic, and intranasal injections.

In certain embodiments, a compound as described herein is administeredin a local rather than systemic manner, for example, via injection ofthe compound directly into an organ, often in a depot preparation orsustained release formulation. In specific embodiments, long actingformulations are administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection.Furthermore, in other embodiments, the drug is delivered in a targeteddrug delivery system, for example, in a liposome coated withorgan-specific antibody. In such embodiments, the liposomes are targetedto and taken up selectively by the organ. In yet other embodiments, thecompound as described herein is provided in the form of a rapid releaseformulation, in the form of an extended release formulation, or in theform of an intermediate release formulation. In yet other embodiments,the compound described herein is administered topically.

Pharmaceutical Composition/Formulation

In some embodiments, the compounds described herein are formulated intopharmaceutical compositions. In specific embodiments, pharmaceuticalcompositions are formulated in a conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen. Any pharmaceuticallyacceptable techniques, carriers, and excipients are used as suitable toformulate the pharmaceutical compositions described herein: Remington:The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: MackPublishing Company, 1995); Hoover, John E., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems,Seventh Ed. (Lippincott Williams & Wilkins1999).

Provided herein are pharmaceutical compositions comprising a compound(i.e., a cyclohexenone compound described herein) and a pharmaceuticallyacceptable diluent(s), excipient(s), or carrier(s). In certainembodiments, the compounds described are administered as pharmaceuticalcompositions in which a compound (i.e., a cyclohexenone compounddescribed herein) is mixed with other active ingredients, as incombination therapy. Encompassed herein are all combinations of activesset forth in the combination therapies section below and throughout thisdisclosure. In specific embodiments, the pharmaceutical compositionsinclude one or more compounds (i.e., a cyclohexenone compound describedherein).

A pharmaceutical composition, as used herein, refers to a mixture of acompound (i.e., a cyclohexenone compound described herein) with otherchemical components, such as carriers, stabilizers, diluents, dispersingagents, suspending agents, thickening agents, and/or excipients. Incertain embodiments, the pharmaceutical composition facilitatesadministration of the compound to an organism. In some embodiments,practicing the methods of treatment or use provided herein,therapeutically effective amounts of compounds (i.e., a cyclohexenonecompound described herein) are administered in a pharmaceuticalcomposition to a mammal having a disease or condition to be treated. Inspecific embodiments, the mammal is a human. In certain embodiments,therapeutically effective amounts vary depending on the severity of thedisease, the age and relative health of the subject, the potency of thecompound used and other factors. The compounds described herein are usedsingly or in combination with one or more therapeutic agents ascomponents of mixtures.

In one embodiment, a compound (i.e., a cyclohexenone compound describedherein) is formulated in an aqueous solution. In specific embodiments,the aqueous solution is selected from, by way of example only, aphysiologically compatible buffer, such as Hank's solution, Ringer'ssolution, or physiological saline buffer. In other embodiments, acompound (i.e., a cyclohexenone compound described herein) is formulatedfor transmucosal administration. In specific embodiments, transmucosalformulations include penetrants that are appropriate to the barrier tobe permeated. In still other embodiments wherein the compounds describedherein are formulated for other parenteral injections, appropriateformulations include aqueous or nonaqueous solutions. In specificembodiments, such solutions include physiologically compatible buffersand/or excipients.

In another embodiment, compounds described herein are formulated fororal administration. Compounds described herein, including a compound(i.e., a cyclohexenone compound described herein), are formulated bycombining the active compounds with, e.g., pharmaceutically acceptablecarriers or excipients. In various embodiments, the compounds describedherein are formulated in oral dosage forms that include, by way ofexample only, tablets, powders, pills, dragees, capsules, liquids, gels,syrups, elixirs, slurries, suspensions and the like.

In certain embodiments, pharmaceutical preparations for oral use areobtained by mixing one or more solid excipients with one or more of thecompounds described herein, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as:for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or otherssuch as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. Inspecific embodiments, disintegrating agents are optionally added.Disintegrating agents include, by way of example only, cross-linkedcroscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or asalt thereof such as sodium alginate.

In one embodiment, dosage forms, such as dragee cores and tablets, areprovided with one or more suitable coating. In specific embodiments,concentrated sugar solutions are used for coating the dosage form. Thesugar solutions, optionally contain additional components, such as byway of example only, gum arabic, talc, polyvinylpyrrolidone, carbopolgel, polyethylene glycol, and/or titanium dioxide, lacquer solutions,and suitable organic solvents or solvent mixtures. Dyestuffs and/orpigments are also optionally added to the coatings for identificationpurposes. Additionally, the dyestuffs and/or pigments are optionallyutilized to characterize different combinations of active compounddoses.

In certain embodiments, therapeutically effective amounts of at leastone of the compounds described herein are formulated into other oraldosage forms. Oral dosage forms include push-fit capsules made ofgelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. In specific embodiments,push-fit capsules contain the active ingredients in admixture with oneor more filler. Fillers include, by way of example only, lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In other embodiments, softcapsules, contain one or more active compound that is dissolved orsuspended in a suitable liquid. Suitable liquids include, by way ofexample only, one or more fatty oil, liquid paraffin, or liquidpolyethylene glycol. In addition, stabilizers are optionally added.

In other embodiments, therapeutically effective amounts of at least oneof the compounds described herein are formulated for buccal orsublingual administration. Formulations suitable for buccal orsublingual administration include, by way of example only, tablets,lozenges, or gels. In still other embodiments, the compounds describedherein are formulated for parental injection, including formulationssuitable for bolus injection or continuous infusion. In specificembodiments, formulations for injection are presented in unit dosageform (e.g., in ampoules) or in multi-dose containers. Preservatives are,optionally, added to the injection formulations. In still otherembodiments, the pharmaceutical compositions of a compound (i.e., acyclohexenone compound described herein) are formulated in a formsuitable for parenteral injection as a sterile suspensions, solutions oremulsions in oily or aqueous vehicles. Parenteral injection formulationsoptionally contain formulatory agents such as suspending, stabilizingand/or dispersing agents. In specific embodiments, pharmaceuticalformulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form. In additional embodiments,suspensions of the active compounds are prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles for usein the pharmaceutical compositions described herein include, by way ofexample only, fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. In certainspecific embodiments, aqueous injection suspensions contain substanceswhich increase the viscosity of the suspension, such as sodiumcarboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension contains suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions. Alternatively, in other embodiments, the activeingredient is in powder form for constitution with a suitable vehicle,e.g., sterile pyrogen-free water, before use.

In one aspect, compounds (i.e., cyclohexenone compounds describedherein) are prepared as solutions for parenteral injection as describedherein or known in the art and administered with an automatic injector.Automatic injectors, such as those disclosed in U.S. Pat. Nos.4,031,893, 5,358,489; 5,540,664; 5,665,071, 5,695,472 and WO/2005/087297(each of which are incorporated herein by reference for such disclosure)are known. In general, all automatic injectors contain a volume ofsolution that includes a compound (i.e., a cyclohexenone compounddescribed herein) to be injected. In general, automatic injectorsinclude a reservoir for holding the solution, which is in fluidcommunication with a needle for delivering the drug, as well as amechanism for automatically deploying the needle, inserting the needleinto the patient and delivering the dose into the patient. Exemplaryinjectors provide about 0.3 mL, 0.6 mL, 1.0 mL or other suitable volumeof solution at about a concentration of 0.5 mg to 50 mg of a compound(i.e., a cyclohexenone compound described herein) per 1 mL of solution.Each injector is capable of delivering only one dose of the compound.

In still other embodiments, the compounds (i.e., cyclohexenone compoundsdescribed herein) are administered topically. The compounds describedherein are formulated into a variety of topically administrablecompositions, such as solutions, suspensions, lotions, gels, pastes,medicated sticks, balms, creams or ointments. Such pharmaceuticalcompositions optionally contain solubilizers, stabilizers, tonicityenhancing agents, buffers and preservatives.

In yet other embodiments, the compounds (i.e., cyclohexenone compoundsdescribed herein) are formulated for transdermal administration. Inspecific embodiments, transdermal formulations employ transdermaldelivery devices and transdermal delivery patches and can be lipophilicemulsions or buffered, aqueous solutions, dissolved and/or dispersed ina polymer or an adhesive. In various embodiments, such patches areconstructed for continuous, pulsatile, or on demand delivery ofpharmaceutical agents. In additional embodiments, the transdermaldelivery of a compound (i.e., a cyclohexenone compound described herein)is accomplished by means of iontophoretic patches and the like. Incertain embodiments, transdermal patches provide controlled delivery ofa compound (i.e., a cyclohexenone compound described herein). Inspecific embodiments, the rate of absorption is slowed by usingrate-controlling membranes or by trapping the compound within a polymermatrix or gel. In alternative embodiments, absorption enhancers are usedto increase absorption. Absorption enhancers or carriers includeabsorbable pharmaceutically acceptable solvents that assist passagethrough the skin. For example, in one embodiment, transdermal devicesare in the form of a bandage comprising a backing member, a reservoircontaining the compound optionally with carriers, optionally a ratecontrolling barrier to deliver the compound to the skin of the host at acontrolled and predetermined rate over a prolonged period of time, andmeans to secure the device to the skin.

Transdermal formulations described herein may be administered using avariety of devices which have been described in the art. For example,such devices include, but are not limited to, U.S. Pat. Nos. 3,598,122,3,598,123, 3,710,795, 3,731,683, 3,742,951, 3,814,097, 3,921,636,3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894, 4,060,084,4,069,307, 4,077,407, 4,201,211, 4,230,105, 4,292,299, 4,292,303,5,336,168, 5,665,378, 5,837,280, 5,869,090, 6,923,983, 6,929,801 and6,946,144.

The transdermal dosage forms described herein may incorporate certainpharmaceutically acceptable excipients which are conventional in theart. In one embodiment, the transdermal formulations described hereininclude at least three components: (1) a formulation of a compound(i.e., a cyclohexenone compound described herein); (2) a penetrationenhancer; and (3) an aqueous adjuvant. In addition, transdermalformulations can include additional components such as, but not limitedto, gelling agents, creams and ointment bases, and the like. In someembodiments, the transdermal formulations further include a woven ornon-woven backing material to enhance absorption and prevent the removalof the transdermal formulation from the skin. In other embodiments, thetransdermal formulations described herein maintain a saturated orsupersaturated state to promote diffusion into the skin.

In other embodiments, the compounds (i.e., cyclohexenone compoundsdescribed herein) are formulated for administration by inhalation.Various forms suitable for administration by inhalation include, but arenot limited to, aerosols, mists or powders. Pharmaceutical compositionsof a compound (i.e., a cyclohexenone compound described herein) areconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Inspecific embodiments, the dosage unit of a pressurized aerosol isdetermined by providing a valve to deliver a metered amount. In certainembodiments, capsules and cartridges of, such as, by way of exampleonly, gelatins for use in an inhaler or insufflator are formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

Intranasal formulations are known in the art and are described in, forexample, U.S. Pat. Nos. 4,476,116, 5,116,817 and 6,391,452, each ofwhich is specifically incorporated herein by reference. Formulations,which include a compound (i.e., a cyclohexenone compound describedherein), which are prepared according to these and other techniqueswell-known in the art are prepared as solutions in saline, employingbenzyl alcohol or other suitable preservatives, fluorocarbons, and/orother solubilizing or dispersing agents known in the art. See, forexample, Ansel, H. C. et al., Pharmaceutical Dosage Forms and DrugDelivery Systems, Sixth Ed. (1995). Preferably these compositions andformulations are prepared with suitable nontoxic pharmaceuticallyacceptable ingredients. These ingredients are found in sources such asREMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st edition, 2005, astandard reference in the field. The choice of suitable carriers ishighly dependent upon the exact nature of the nasal dosage form desired,e.g., solutions, suspensions, ointments, or gels. Nasal dosage formsgenerally contain large amounts of water in addition to the activeingredient. Minor amounts of other ingredients such as pH adjusters,emulsifiers or dispersing agents, preservatives, surfactants, gellingagents, or buffering and other stabilizing and solubilizing agents mayalso be present. Preferably, the nasal dosage form should be isotonicwith nasal secretions.

For administration by inhalation, the compounds described herein, may bein a form as an aerosol, a mist or a powder. Pharmaceutical compositionsdescribed herein are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, such as, by way of example only, gelatin foruse in an inhaler or insufflator may be formulated containing a powdermix of the compound described herein and a suitable powder base such aslactose or starch.

In still other embodiments, the compounds (i.e., cyclohexenone compoundsdescribed herein) are formulated in rectal compositions such as enemas,rectal gels, rectal foams, rectal aerosols, suppositories, jellysuppositories, or retention enemas, containing conventional suppositorybases such as cocoa butter or other glycerides, as well as syntheticpolymers such as polyvinylpyrrolidone, PEG, and the like. In suppositoryforms of the compositions, a low-melting wax such as, but not limitedto, a mixture of fatty acid glycerides, optionally in combination withcocoa butter is first melted.

In certain embodiments, pharmaceutical compositions are formulated inany conventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen. Any pharmaceutically acceptable techniques,carriers, and excipients is optionally used as suitable and asunderstood in the art. Pharmaceutical compositions comprising a compound(i.e., a cyclohexenone compound described herein) may be manufactured ina conventional manner, such as, by way of example only, by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or compression processes.

Pharmaceutical compositions include at least one pharmaceuticallyacceptable carrier, diluent or excipient and at least one compound(i.e., cyclohexenone compounds described herein) described herein as anactive ingredient. The active ingredient is in free-acid or free-baseform, or in a pharmaceutically acceptable salt form. In addition, themethods and pharmaceutical compositions described herein include the usecrystalline forms (also known as polymorphs), as well as activemetabolites of these compounds having the same type of activity. Alltautomers of the compounds described herein are included within thescope of the compounds presented herein. Additionally, the compoundsdescribed herein encompass unsolvated as well as solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike. The solvated forms of the compounds presented herein are alsoconsidered to be disclosed herein. In addition, the pharmaceuticalcompositions optionally include other medicinal or pharmaceuticalagents, carriers, adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure, buffers, and/or other therapeutically valuable substances.

Methods for the preparation of compositions comprising the compoundsdescribed herein include formulating the compounds with one or moreinert, pharmaceutically acceptable excipients or carriers to form asolid, semi-solid or liquid. Solid compositions include, but are notlimited to, powders, tablets, dispersible granules, capsules, cachets,and suppositories. Liquid compositions include solutions in which acompound is dissolved, emulsions comprising a compound, or a solutioncontaining liposomes, micelles, or nanoparticles comprising a compoundas disclosed herein. Semi-solid compositions include, but are notlimited to, gels, suspensions and creams. The form of the pharmaceuticalcompositions described herein include liquid solutions or suspensions,solid forms suitable for solution or suspension in a liquid prior touse, or as emulsions. These compositions also optionally contain minoramounts of nontoxic, auxiliary substances, such as wetting oremulsifying agents, pH buffering agents, and so forth.

In some embodiments, pharmaceutical composition comprising at leastcompound (i.e., cyclohexenone compounds described herein) illustrativelytakes the form of a liquid where the agents are present in solution, insuspension or both. Typically when the composition is administered as asolution or suspension a first portion of the agent is present insolution and a second portion of the agent is present in particulateform, in suspension in a liquid matrix. In some embodiments, a liquidcomposition includes a gel formulation. In other embodiments, the liquidcomposition is aqueous.

In certain embodiments, pharmaceutical aqueous suspensions include oneor more polymers as suspending agents. Polymers include water-solublepolymers such as cellulosic polymers, e.g., hydroxypropylmethylcellulose, and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers. Certain pharmaceutical compositionsdescribed herein include a mucoadhesive polymer, selected from, forexample, carboxymethylcellulose, carbomer (acrylic acid polymer),poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylicacid/butyl acrylate copolymer, sodium alginate and dextran.

Pharmaceutical compositions also, optionally include solubilizing agentsto aid in the solubility of a compound (i.e., cyclohexenone compoundsdescribed herein). The term “solubilizing agent” generally includesagents that result in formation of a micellar solution or a truesolution of the agent. Certain acceptable nonionic surfactants, forexample polysorbate 80, are useful as solubilizing agents, as canophthalmically acceptable glycols, polyglycols, e.g., polyethyleneglycol 400, and glycol ethers.

Furthermore, pharmaceutical compositions optionally include one or morepH adjusting agents or buffering agents, including acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

Additionally, pharmaceutical compositions optionally include one or moresalts in an amount required to bring osmolality of the composition intoan acceptable range. Such salts include those having sodium, potassiumor ammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

Other pharmaceutical compositions optionally include one or morepreservatives to inhibit microbial activity. Suitable preservativesinclude mercury-containing substances such as merfen and thiomersal;stabilized chlorine dioxide; and quaternary ammonium compounds such asbenzalkonium chloride, cetyltrimethylammonium bromide andcetylpyridinium chloride.

Still other pharmaceutical compositions include one or more surfactantsto enhance physical stability or for other purposes. Suitable nonionicsurfactants include polyoxyethylene fatty acid glycerides and vegetableoils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40.

Still other pharmaceutical compositions may include one or moreantioxidants to enhance chemical stability where required. Suitableantioxidants include, by way of example only, ascorbic acid and sodiummetabisulfite.

In certain embodiments, pharmaceutical aqueous suspension compositionsare packaged in single-dose non-reclosable containers. Alternatively,multiple-dose reclosable containers are used, in which case it istypical to include a preservative in the composition.

In alternative embodiments, other delivery systems for hydrophobicpharmaceutical compounds are employed. Liposomes and emulsions areexamples of delivery vehicles or carriers herein. In certainembodiments, organic solvents such as N-methylpyrrolidone are alsoemployed. In additional embodiments, the compounds described herein aredelivered using a sustained-release system, such as semipermeablematrices of solid hydrophobic polymers containing the therapeutic agent.Various sustained-release materials are useful herein. In someembodiments, sustained-release capsules release the compounds for a fewhours up to over 24 hours. Depending on the chemical nature and thebiological stability of the therapeutic reagent, additional strategiesfor protein stabilization may be employed.

In certain embodiments, the formulations described herein include one ormore antioxidants, metal chelating agents, thiol containing compoundsand/or other general stabilizing agents. Examples of such stabilizingagents, include, but are not limited to: (a) about 0.5% to about 2% w/vglycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% toabout 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e)about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/vpolysorbate 80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h)arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l)pentosan polysulfate and other heparinoids, (m) divalent cations such asmagnesium and zinc; or (n) combinations thereof.

EXAMPLE Example 1 Preparation of the Exemplary Compound 1

The exemplary Compound 1 (i.e., Antroq) was isolated from thesolid-state fermented mycelium of Antrodia camphorata followed the knownmethod. (Lee, et al., Planta Med, 73: 1412-1415, 2007) Compound 1 iseffective at a range of 10-50 mg/kg body weight, based on a previousstudy and empirical use. (Chang, et al., Evid Based Complement AlternatMed, 2008) Unless indicating otherwise, 50 mg/kg body weight of Compound1 was used as a dosage in the following experiments.

Alternatively, the exemplary Compound 1 may be prepared from4-hydroxy-2,3-dimethoxy-6-methylcyclohexa-2,5-dienone or the like.Similarly, other cyclohexenone compounds having the structure

are isolated from Antrodia camphorata or prepared synthetically orsemi-synthetically from the appropriate starting materials. An ordinaryskilled in the art would readily utilize appropriate conditions for suchsynthesis.

Example 2 Establishment of the FSGS Model and Experimental Protocol

Experiments described herein were performed on 8-week-old female BALB/cmice. The FSGS mice were injected intravenously with a single dose ofadriamycin (0.1 mg/10 g body weight). The mice were given Compound 1 sixhours before adriamycin injection by gavage daily till sacrifice. BALB/cmice intraperitoneal injected with normal saline were used as normalcontrols, while ASLN mice given vehicle (corn oil) by gavage were usedas disease controls. Mice were killed at day 7, 14, or 21 after diseaseinduction, and spleen, renal cortical tissue, and blood samplescollected and stored appropriately until analysis. All animalexperiments were performed with the ethical approval of theInstitutional Animal Care and Use Committee of The National DefenseMedical Center, Taiwan and performed according to the ethical rules inthe NIH Guide for the Care and Use of Laboratory Animals.

Clinical and Renal Function Evaluation

Urine samples were collected in metabolic cages at day 3, 7, 14, and 21,and urine protein was determined as described previously. (Shui, et al.,Nephrol Dial Transplant, 21: 1794-1802, 2006) Serum samples werecollected at day 7, 14, or 21 when mice were sacrificed to measure serumlevels of BUN and Cr.

Pathologic Evaluation

Formalin-fixed and paraffin-embedded renal sections were prepared asdescribed in Shui et al. for renal pathologic evaluation. (Shui, et al.,Transl Res, 150: 216-222, 2007) Renal pathology and scoring of renallesions were performed according to the known method disclosed in Shui.For evaluations of EPHL and sclerosis, at least 50 glomeruli in renaltissue sections for each case were examined. The number of glomeruliwith EPHLs was expressed as a percentage of the total number ofevaluated glomeruli according to methods disclosed before (see forexample, Shui, et al., Nephrol Dial Transplant, 21: 1794-1802, 2006; Ka,et al., Nephrol Dial Transplant, 21: 288-298, 2006).

For IHC, formalin-fixed and paraffin-embedded renal sections wereprepared and incubated with primary antibodies against mouse Desmin (LabVision, CA, USA), CD3 (pan-T cell; Serotec, NC, USA), F4/80(monocytes/macrophages; Serotec), IL-6 (R&D Systems, MN, USA), NF-κB p65(Cell Signaling Technology, MA, USA), collagen I, III, and IV (SouthernBiotech, AL, USA), or TGF-β1 (Santa Cruz Biotechnology, CA, USA),biotinylated second antibodies (Dako, Glostrup, Denmark), andavidin-biotin-peroxidase complex (Dako). Semi-quantitative evaluation ofstaining was performed.

Example 3 ROS and NO Determination

Kidney in-situ superoxide anion production was determined by DHElabeling according to the known method. (Wu, et al., Nephrol DialTransplant, 2008, 23: 3082-3090, or Ka, et al., J. Am. Soc. Nephrol.2007, 18:2473-2485) Fluorescent images were quantified by counting thepercentage of positive nuclei in the total nuclei per kidney crosssection. Sera and kidney tissues were assessed for superoxide anionaccording to the known method. (Wu, et al., J Pineal Res, 2001, 30:147-156, or Ka, et al., J. Am. Soc. Nephrol. 2007, 18:2473-2485) For ROSlevels of serum, urine, and renal tissue, the samples were incubatedwith Krebs-HEPES buffer, and lucigenin (Sigma-Aldrich Chemical Co, MO)at 1.25 mM was used as substrate. Luminescence counts were obtained induplicate at 15 sec intervals by a microplate luminometer (HidexMicroplate Luminometer, Finland), as previously described (Kretzler, etal., Virchows Arch, 425: 181-193, 1994). The superoxide anion activitywas expressed as relative luminescence units (RLU) per 15 min per mg oforgan dry weight (i.e., RUL/15 min/mg) or RLU/15 min/ml.

NO levels in serum were detected with NO Detection kit (iNtRONBiotechnology, Seongnam, Korea), based on diazotization (Griess method),according to the manufacturer's instructions.

Example 4 Measurement of Cellular GPx Activity in the Kidney

GPx activity in renal tissues was measured using a commercialGlutathione Peroxidase Assay kit (Cayman, MI, USA) according to themanufacturer's instructions. Enzyme activity was expressed relative tothe protein concentration of glomerular homogenates.

Example 5 Western Blot Analysis of Nrf2 and p47^(phox)

The preparation of cytoplasmic and nuclear proteins of renal tissues wasextracted using the Nuclear Extract Kit (Active Motif, Tokyo, Japan)according to the manufacturer's instructions. Target proteins in thecytoplasmic and/or nuclear fractions of kidney tissue were measured byWestern blot analysis using rabbit antibodies against mouse Nrf2, orp47^(phox) (Santa Cruz). Antibodies to histone H3 (Cell Signaling, CO,USA) and β-actin (Santa Cruz) were used for measuring the housekeepingproteins for nuclear and cytosolic target proteins, respectively.

Example 6 Measurement of TGF-β1

The TGF-β1 protein levels in serum and renal tissue were measured usingthe commercial ELISA kits (R&D Systems), according to the manufacturer'sinstructions. Samples were acidified with 1 N HCl and neutralized with1.2 N NaOH/0.5 M HEPES to assay for the amount of TGF-β1.

Example 7 AcP-IgAN Model in B Cell-Deficient Mice

B cell-deficient mice (B6.12952-Igh-6tmlCgn/J) were obtained from theAcademia Sinica (Professor John T. Kung, Institute of MolecularBiology), and maintained at the animal center of the National DefenseMedical Center, Taipei, Taiwan. AcP-IgAN was induced in the mice bydaily injection of purified IgA anti-phosphorylcholine antibodies andpneumococcal C-polysaccharide (PnC) as described previously (Chao, etal., Kidney Int. 70:283-297; 2006). All animal experiments wereperformed with the approval of the Institutional Animal Care and UseCommittee of The National Defense Medical Center, Taiwan, and wereconsistent with the NIH Guide for the Care and Use of LaboratoryAnimals.

Clinical and Pathological Evaluation

Body weight of the mice was measured weekly. Urine samples werecollected in metabolic cages weekly, and urine protein determinedfollowed the know methods (Chao, et al., Kidney Int. 70:283-297; 2006).Serum samples were collected at day 3 and day 28 to measure serum levelsof blood urea nitrogen (BUN) and creatinine (Cr).

For renal histopathology, the tissues were fixed in 10% bufferedformalin and embedded in paraffin, and then sections (4 μm) wereprepared and stained with hematoxylin and eosin (H&E). The proportion ofglomeruli showing proliferation, crescent formation, sclerosis, orperi-glomerular inflammation was counted in 50 randomly sampledglomeruli by light microscopy at the magnification of 400×.

Example 8 Immunofluorescence (IF), Immunohistochemistry (IHC), andDetection of Apoptosis

For IF, frozen renal tissues were prepared as described previously andincubated with fluorescein isothiocyanate (FITC)-conjugated goatanti-mouse IgA or C3 antibodies (Cappel, NC). Scoring of stainingintensity was performed as described in Chao, et al., Kidney Int.70:283-297 (2006).

For IHC, formalin fixed and paraffin embedded tissue sections or frozensections were incubated with against IL-6 (R&D Systems, MN), MCP-1(Santa Cruz, Calif.), F4/80 (monocytes/macrophages; Serotec, NC),collagen IV (Southern Biotech, AL), TGF-β1 (Santa Cruz), phopsho-NFκBp65 (Cell Signaling, MA), CD3 (pan-T cell; Serotec), CD4 (T helper cell;BioLegend, CA), CD8 (cytotoxic T cell), CD11b (macrophages/neutrophils),or CD11c (dendritic cells) (BD Biosciences, CA) antibodies. TheFITC-conjugated, Alexa Fluor 488-conjugated (Invitrogen, CA) orhorseradish peroxidase (HRP) conjugated secondary antibodies (DAKO,Denmark) were then applied to the sections. Hematoxylin or4′,6-diamidino-2-phenylindole (DAKO) was used in the counter stainingfor nuclei.

For the detection of apoptosis, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) was employed.Formalin-fixed and paraffin-embedded tissue sections were stained withApopTag plus peroxidase in situ apoptosis detection kit (Chemicon, CA)according to the manufacturer's instructions. For scoring, the corticalrenal area (including glomerular and peri-glomerular areas) wereexamined and expressed as cells/glomerular cross-section.

Example 9 Flow Cytometry

Splenocytes from the mice were treated with Tris-buffered ammoniumchloride to eliminate erythrocytes, washed, resuspended in RPMI 1640supplemented with 10% fetal calf serum, HEPES buffer, L-glutamine, andpenicillin/streptomycin (all from Invitrogen). The cells were stainedwith either surface marker for T or B cell activation. FITC-conjugatedanti-mouse CD3, CD4, CD8, or CD19 (B cell) antibodies and phycoerythrin(PE)-conjugated anti-mouse CD69 antibodies (all from BD Biosciences)were analyzed with FACSCalibur (BD Biosciences).

Example 10 T Cell Proliferation Analysis

Splenocytes from the mice were prepared as described as above, then werecultured in triplicate in wells (5×105 cells in 200 μl/well) in 96-wellflat-bottomed microtiter plates previously coated overnight at 4° C.with 0.25 μg/ml of anti-mouse CD3 antibody (BD Biosciences). After 48hr, the cultures were pulsed with 1 μCi of 3H-methyl thymidine (AmershamPharmacia Biotech, NJ), harvested 16 hr later, and the incorporated3H-methyl thymidine measured using a TopCount (Packard, PerkinElmer,MA).

Example 11 Enzyme-Linked Immunosorbent Assay (ELISA) of IL-1β, IL-6,IL-18 and MCP-1

Serum levels of IL-1β (eBioscience, CA), IL-6 (eBiosceience), IL-18(MBL, Japan), and MCP-1 (eBiosceience) were measured using commercialELISA kits according to the manufacturer's instructions.

Nuclear proteins were extracted using a nuclear extract kit (ActiveMotif, Japan). Phospho-NF-κB p65 and nuclear factor-erythroid-2-relatedfactor 2 (NrF2) were measured in renal tissue nuclear protein extractsusing Trans-AM ELISA assay kits (Active Motif), according to themanufacturer's instructions. Renal cytosolic protein extracts using RIPAbuffer (Cell signaling). Cytosolic glutathione peroxidase (GPx) (Cayman,MI) and cytosolic heme oxygenase-1 (HO-1) (Enzo Life Sciences, NY) weremeasured using commercial ELISA kits according to the manufacturer'sinstructions. Both were expressed relative to the protein concentrationin the lysate.

In all ELISAs, the absorbance at 450 nm was measured using an ELISAplate reader (Bio-Tek, MA).

Example 12 Real-Time PCR Analysis

Total cortical kidney RNA was extracted with TriZOL reagents(Invitrogen) from cortical tissue of the kidney. For first-strand cDNAsynthesis, 1.5 μg of total RNA was used in a single-round reversetranscriptase reaction. The reaction mixture consisted of 0.9 μl ofOligo (dT) 12 to 18 primer, 1.0 mM deoxyribonucleotide triphosphate(dNTP), 1 μl first strand buffer, 0.4 mM dithiothreitol, 80 U ofRNaseout recombinant ribonuclease inhibitor, and 300 U of superscript IIRNase H (Invitrogen). Real-time PCR was performed on an ABI Prism 7700Sequence Detection System (Applied Biosystems, CA). All of the probesand primers were Assays-on-Demand Gene expression products (AppliedBiosystems). Real-time PCR reactions were using 10 μl of cDNA, 12.5 μlof TaqMan Universal PCR Master Mix (Applied Biosystems), and 1.25 μl ofthe specific probe/primer mixed in a total volume of 25 μl. The thermalcycler conditions were as follows: 2 min at 50° C., 10 min at 95° C., 40cycles of denaturation (15 s at 95° C.), and combinedannealing/extension (1 min at 60° C.).

Example 13 Western Blot Analysis

Each protein sample was run on a 10% SDS-PAGE gel. The gel waselectroblotted onto polyvinylidene difluoride nitrocellulose membrane(Amersham Int., UK); incubated for 1 h in blocking buffer (Tris-bufferedsaline that contained 5% skim milk); and incubated with rabbit againstnacht domain-, leucine-rich repeat-, and pyrin domain (PYD)-containingprotein 3 (NLRP3), caspase-1 or β-actin (all from Santa Cruz) antibodiesat 4° C. overnight. After washing, the membrane was incubated withHRP-conjugated goat anti-rabbit (DAKO) antibody for 1 h at roomtemperature. The membrane-bound antibody detected was incubated withchemiluminescent reagent plus (PerkinElmer Life Sciences, MA) andcaptured on x-ray film.

Example 14 Data Analysis

The results are presented as the mean±SEM. Comparisons between twogroups were performed using Student's t test. Differences among multiplegroups were determined with the one-way analysis of variance (ANOVA)using Tukey's method for post hoc analysis. A p value<0.05 wasconsidered statistically significant.

FSGS Model

As a disease control, FSGS mice treated with vehicle (i.e., control FSGSmice) showed increased urine protein levels from day 7 of the treatmentand continued to rise up to the end of the study at day 21 (See FIG.1A). This effect was greatly suppressed in Compound 1 treated FSGS mice(FSGS+Antroq mice) where the urine protein levels were similar to thosein normal control mice. In addition, compared to control FSGS mice,which showed a significant and persistent increase serum levels of bloodurea nitrogen (BUN) (FIG. 1B) and creatinine (Cr) (FIG. 1C) from day 14to day 21, FSGS+Antroq (i.e. Compound 1) mice exhibited much betterrenal function. There was no significant difference in the levels of BUNor Cr levels among the normal control, control FSGS, and FSGS+Antroqmice on day 7.

Histopathological examination was performed on kidney sections atvarious time-points (FIG. 2A). In control FSGS mice, expansion of theextracellular matrix and deposition of hyaline mass in the glomeruliwere occasionally seen on day 7 and these became obvious scleroticlesions from day 14 to day 21, compared to normal control mice.Importantly, the mice exhibited a significant and steady increase in thepercentage of glomeruli containing EPHL and peri-glomerular mononuclearleukocyte infiltration from day 14 to day 21, suggesting a progressivepathological status. In contrast, these progressive renal lesions weregreatly reduced in FSGS+Antroq mice. Besides, podocyte injury and losshave been proposed as critical pathogenic events in the development ofFSGS. To assess changes in podocyte phenotype during the Antroqtreatment of FSGS, we studied the expression of desmin, apodocyte-specific marker by immunohistochemistry (IHC). As shown in FIG.2B, FSGS+Antroq mice showed significantly reduced expression of desmincompared to control FSGS mice on both days 14 and 21, although milddesmin expression levels were observed compared to normal control miceon day 21.

These results show that the exemplary Compound 1 improves proteinuria,renal function, and renal lesions including epithelial hyperplasialesion (EPHL), a severe index of glomerular injury.

Systemic Suppression of Oxidative Stress in Serum and Urine

The serum superoxide anion levels were significantly increased incontrol FSGS mice (FSGS+vehicle) compared with normal control mice onday 7 and day 14, and then slightly dropped on day 21, although stillhigher than normal control mice. Administration of the exemplaryCompound 1 (Antroq) effectively attenuated the levels of superoxideanion to a level similar to that observed in normal control mice fromday 7 to day 21 (FIG. 3A). In addition, serum NO levels in control FSGSmice were significantly increased on day 7 and remained high levels onday 14 and day 21. The high serum NO levels were significantlysuppressed when the exemplary Compound 1 was administered to the FSGSmice (FSGS+Antroq) (FIG. 3B). The superoxide anion levels in urinesignificantly increased on day 7 and remained to day 21 in FSGS controlmice, compared to normal control mice. In contrast, administration ofthe exemplary Compound 1 significantly lowered the levels of superoxideanion in FSGS+Antroq mice (FIG. 3C). Although urine NO levels in controlFSGS mice were significantly increased from day 14 to day 21, againadministration of the exemplary Compound 1 effectively blunted theseeffects in FSGS+Antroq mice (FIG. 3D). There was no detectabledifference in urine levels of NO among normal control, FSGS+vehicle, andFSGS+antroq mice at day 7.

Local Inhibition of ROS Production in Kidney Tissues

As shown in FIG. 3E, the superoxide anion levels in the kidney ofcontrol FSGS mice were significantly increased on day 14 and continuedto rise up to day 21, compared with normal control mice. Antroqadministration effectively attenuated the levels of superoxide anion toa level similar to that observed in normal control mice on both day 14and day 21.

To further localize ROS production in the kidney, in-situ ROS productionin renal tissue was analyzed by using the dihydroethidium (DHE) assay.As shown in FIG. 3F, DHE fluorescence was significantly increased in thekidney, mainly in glomeruli and some renal tubules, of control FSGS micefrom day 14 to day 21, showing increased in-situ ROS production comparedto normal mice. In contrast, only very low DHE fluorescence was observedin FSGS+Antroq mice at both day 14 and day 21.

Nrf2-Mediated Antioxidant Signaling Pathway

NAD(P)H oxidase subunit p47^(phox) protein expression levels, Nrf2translocation into nuclei (activation), and GPx activity in the kidneywere further measured to determine the effects of Antroq on antioxidantsignaling pathway.

As shown in FIGS. 4A and C, protein levels of NAD(P)H oxidase subunitp47^(phox) were significantly increased in control FSGS mice from day 14to day 21 and these effects were abolished by administration of Antroq.In contrast, FSGS+Antroq mice showed strongly augmented Nrf2translocation into nuclei compared to control FSGS mice or normalcontrols from day 14 and this remained high on day 21 (FIGS. 4B and D).

In addition, as shown in FIG. 4E, marked decreased of activity of GPx,one of the phase II enzymes downstream of Nrf2, was seen in control FSGSmice from day 7 to day 21, compared to normal control mice. However,compared with control FSGS mice, FSGS+Antroq mice showed recovered GPxactivity in the kidney on day 7 and maintained until day 21 when themice were sacrificed.

These results show the exemplary Compound 1 inhibits ROS/NO production,but enhances Nrf2 activation and GPx activity.

T Cell and Macrophage Infiltration

The interstitial recruitment of macrophages and lymphocytes as majorsource of inflammatory and profibrotic mediators plays an important rolein progression of FSGS.^(1, 38) The effects of administering theexemplary Compound 1 on T cells and/or monocytes/macrophagesinfiltration in kidney were next assessed. Compared to control FSGS micewhich showed that a profound T cells (CD3⁺) and monocytes/macrophages(F4/80⁺) infiltration was noted in the periglomerular region of therenal interstitium on day 14 and day 21, FSGS+Antroq mice showed thesimilar pattern to normal control mice (FIGS. 5A and B). IL-6 production

The production of IL-6 in the kidney was further measured. As shown byIHC in FIG. 6A, the protein expression of IL-6 were significantlyincreased as early as day 7 and continued to rise up to day 21 till micewere sacrificed. However, FSGS+Antroq mice showed significantly reducedrenal protein expression levels of and IL-6, compared with control FSGSmice.

Blocking of NE-κB Activation

The effects of the exemplary Compound 1 administration on NF-κBactivation in kidney tissues were investigated. As shown in FIG. 6B,compared to control FSGS mice showed significantly stimulated thenuclear NF-κB p65 expression on day 14 and day 21, NF-κB activation inFSGS+Antroq mice were markedly inhibited. Consistent with the IHC, ELISAassay for renal tissue nuclear protein extracts also demonstrated thatnuclear protein expression of NF-κB p65 was tend to increased on day 14and significantly increased on day 21 in control FSGS mice compared tonormal control mice. These effects was slightly blocked by Compound 1(Antroq) administration on day 14 and significantly inhibited on day 21in FSGS+Antroq mice. There was no significant difference in eithercontrol FSGS mice or FSGS+Antroq mice early on day 7 (FIG. 6C).

Thus, the blocking of NF-κB activation in the kidney provides beneficialeffects on inflammation when the cyclohexenone compound provided hereinis administered.

The effects of administration of the cyclohexenone compound providedherein on glomerulosclerosis in the FSGS model were further evaluated,focusing on fibrosis related proteins, collagen I, III, and IV. As shownin FIGS. 7A-C, IHC demonstrated marked renal expression of collagen Iand IV from day 14 to day 21, and collagen III on day 21 in control FSGSmice compared with normal control mice and that Compound 1administration was associated with significant suppression of theexpression of these proteins, their levels in FSGS+Antroq mice not beingsignificantly different from those in normal control mice. TGF-β1 is afundamental growth factor and cytokine in renal fibrosis and creascentformation. ELISA showed that compared with normal control mice, TGF-β1protein in serum (FIG. 8A) and kidney tissues (FIG. 8B) weresignificantly upregulated on day 14 and dramatically increased on day 21in control FSGS mice. However, administration of the exemplary Compound1 greatly abrogated the increased TGF-β1 protein levels in both serumand kidney. IHC was further performed to determine the changes inexpression levels of TGF-β1 protein in renal tissues. Similarly,FSGS+Antroq mice showed a significantly lower TGF-β1 protein expressionin the kidney than control FSGS mice as demonstrated by IHC (FIG. 8C).As such, inhibition of TGF-β1 expression provides the beneficial effectson the treatment of glomerulosclerosis when the cyclohexenone compoundprovided herein is used.

IgAN Model

As a disease control, AcP-IgAN mice treated with vehicle (controlAcP-IgAN mice) showed increased urine protein levels from day 7 ofdisease induction and these continued to rise up to the end of the studyat day 28 (FIG. 9). This effect was greatly suppressed in AcP-IgAN micetreated with Compound 1 (AcP-IgAN+Antroq mice), although they stillshowed mild proteinuria compared to normal controls. In addition,compared to control AcP-IgAN mice, which showed significantly increasedserum levels of BUN (FIG. 9B) and creatinine (FIG. 9C) at day 28,AcP-IgAN+Antroq mice revealed much better renal function, although therewas no significant difference in serum levels of BUN and Cr betweencontrol AcP-IgAN, AcP-IgAN+Antroq and normal control mice at day 3.

The body weight of mice was recorded every week. The growth of controlAcP-IgAN and AcP-IgAN+Antroq mice was no different from that of normalcontrols. In addition, all mice of either group showed normal activityand no evidence of hair loss or appetite change.

Renal Pathology

As shown in FIGS. 9A-E, at day 28, control AcP-IgAN mice developed adiffuse proliferation associated with neutrophil infiltration, focal buttypical crescents, and/or segmental sclerosis in the glomerulus, withintense peri-glomerular mononuclear leukocyte infiltration and scatteredtubular atrophy associated with protein casts, suggesting an aggressiveand exacerbated status of the diseased kidney in comparison with thereported glomerular (Lai, K. N. Nephron. 92:263-270 (2002) ; Lai, etal., Nephron. 69:1-8 (1995); Chen, et al., J. Clin. Lab. Analysis.6:35-39 (1992); Kashem, et al., Kidney Int. 45: 868-875 (1994)) andinterstitial (Falk, et al., Kidney Int. 47:177-185 (1995); van Es, etal., Kidney Int. 73:1426-1433 (2008); Tones, et al., Kidney Int.73:327-333 (2008); Walsh, et al., Clin J Am Soc. Nephrol. 5:425-430(2010); Fujinaka, et al., J. Nephrol. 20:357-363 (2007)) histopathologyin the kidney. Except for the renal lesions from the glomerular immunedeposits, all of these renal lesions were substantially inhibited in theAcP-IgAN+Antroq mice. At day 3, control AcP-IgAN mice started to developfocal, but this renal histopathology was again significantly inhibitedby Compound 1 administration in AcP-IgAN+Antroq mice.

Renal Fibrosis-Related Gene and Protein Expression

Detection of both mRNA and protein levels of TGF-β1 and Col-IV wereperformed in the mice. Although control AcP-IgAN mice showed greatlyenhanced mRNA expression of TGF-β1 and Col-IV, respectively, in thekidney at day 28, compared to normal controls, these two effects weregreatly inhibited by Compound 1 administration in AcP-IgAN+Antroq mice(FIGS. 10A-D). Only basal mRNA levels of the two fibrosis-related geneswere observed in the two groups of mice at day 3. In parallel, bothTGF-beta and Col-IV protein levels were greatly increased in controlAcP-IgAN mice at day 28, but this effect was substantially inhibited inAcP-IgAN+Antroq mice, as demonstrated by IHC.

Cell Immunity and Renal Inflammation

Cell mediated immunity has long been implicated in the pathogenesis ofIgAN. Flow cytometry in splenocytes was performed to identify theactivation of CD3, CD4 and CD8, respectively. As shown in FIGS. 11A-C,an noticeable increase in percentage of CD3⁺/CD69⁺, CD4⁺/CD69⁺ orCD8⁺/CD69⁺ cells was observed in control AcP-IgAN mice at as early asday 3, compared to normal controls, although there was no such effectfor all subtypes of T cells later at day 28. In contrast, Compound 1administration induced a significant reduction in percentage ofCD3⁺/CD69⁺ T cells in AcP-IgAN+Antroq mice at day 3, compared to that ofcontrol AcP-IgAN mice. There was no significant difference in thepercentage of either CD4⁺/CD69⁺ or CD8⁺/CD69⁺ T cells between controlAcP-IgAN and AcP-IgAN+Antroq mice at day 3. The percentage of each ofall the three subtypes of T cells was no different from that of normalcontrols at day 28. As demonstrated by thymidine uptake analysis,AcP-IgAN mice revealed a greatly increased proliferation of CD3⁺T cellin splenocytes compared to that of AcP-IgAN+Antroq mice early at day 3(FIGS. 11A-C), and either group of mice showed near baseline levels ofproliferation similar to normal controls later at day 28. In parallel,IHC was performed to evaluate the phenotypic expression of mononuclearleukocytes that infiltrated in the kidney of the mice. As shown in FIGS.12A-F, focal but intense CD3⁺ (pan-T) cells (FIGS. 12A-C), CD4⁺ Thcells, CD8⁺ Tc cells, CD11c+neutrophils, F4/80+monocytes/macrophages,and CD11b+monocytes/macrophages (FIGS. 12D-F) were identified in therenal interstitial tissue, mostly in a peri-glomerular pattern incontrol AcP-IgAN mice at day 28, compared to normal controls, althoughonly very few inflammatory cells were seen in the kidney at day 3. Incontrast, AcP-IgAN+Antroq mice showed significantly decreasedinfiltration of such inflammatory cells in the kidney, compared toAcP-IgAN mice, at day 28, and there was no detectable signals suggestinginfiltration of pan-T cells, neutorphils, and monocytes/macrophages inthe kidney of AcP-IgAN+Antroq mice at day 3.

Oxidative Stress, Nrf2 and Related Pathway

ROS has been considered a major detrimental chemical mediator toacceleration and deterioration in various types of renal disorders,including IgAN. The expression levels of ROS were detected systemicallyin blood and locally in renal tissues. The AcP-IgAN mice showed greatlyelevated ROS levels in serum at both day 3 and day 28, and in urine andrenal tissues at day 28, compared to normal controls (FIGS. 13A-F). Incontrast, the exemplary invention Compound 1 administration caused asubstantial reduction in ROS levels in sera at as early as day 3, and insera, urine and renal tissues of AcP-IgAN+Antroq mice later at day 28,compared to those of control AcP-IgAN mice. In addition, controlAcP-IgAN mice showed significantly higher urine nitric oxide (NO) levelsthan normal controls, at as early as day 3 until day 28. However,AcP-IgAN+Antroq mice showed significantly decreased urine NO levels atboth day 3 and day 28, compared to control AcP-IgAN mice. Although therewas no significant difference in serum levels of NO at day 3 betweencontrol AcP-IgAN and AcP-IgAN+Antroq mice, substantially reduced serumlevels of NO were observed at day 28 in AcP-IgAN+Antroq mice, comparedto control AcP-IgAN mice.

The potential mechanistic events that might be involved in thesefindings were further investigated by the administration of an exemplaryinvention Compound 1. As shown in FIGS. 14A-F, the expression levels ofboth mRNA and protein of Nrf2 were found greatly increased inAcP-IgAN+Antroq mice, compared to those of control AcP-IgAN miceassociated with near normal baseline levels of both mRNA and protein,starting at as early as day 3 until day 28 when the animals weresacrificed. In addition, glutathione peroxidase (GPx), a downstreamprotein of Nrf2, was found to have significantly higher expressionlevels in renal tissues of AcP-IgAN+Antroq mice at day 28, compared tocontrol AcP-IgAN mice, while there was no difference at day 3 betweenthe two groups of mice.

Serum Levels of Pro-Inflammatory Cytokines

First, as shown in FIG. 15B, serum levels of MCP-1 were significantlyelevated at as early as day 3 in control AcP-IgAN mice, and this effectcontinued to augment until day 28 when the animals were sacrificed. Incontrast, this effect was greatly inhibited in AcP-IgAN+Antroq mice,showing only baseline levels. In addition, although there was nodifference in serum IL-6 levels between control AcP-IgAN,AcP-IgAN+Antroq and normal control mice at day 3, the AcP-IgAN+Antroqmice showed substantially decreased serum levels of IL-6 at day 28 (FIG.15A), compared to control AcP-IgAN mice which showed significantlyincreased serum IL-6 levels compared to normal controls. At day 28,control AcP-IgAN mice showed significantly increased serum levels ofIL-1β compared to normal controls, but this effect was greatly inhibitedin AcP-IgAN+Antroq mice (FIG. 15C). At day 3, there was no detectableincrease in IL-18 levels in both control AcP-IgAN and AcP-IgAN+Antroqmice compared to normal controls (FIG. 15D). At day 28, significantlyelevated serum levels of IL-18 were observed in control AcP-IgAN mice,but there was a substantial inhibition in serum IL-18 levels inAcP-IgAN+Antroq mice, although at day 3 both groups of mice hadsimilarly elevated serum IL-18 levels compared to normal controls.

NLRP3 Inflammasome Activation (in the Kidney)

Increasing evidence supports NACHT, LRR and PYD domains-containingprotein 3 (NALP3) inflammasome to be an active and crucial player in theinnate immune response and link to the adaptive immunity. Although therole of NLRP3 in host response to pathogen associated molecules is welldocumented, its role in immune complex-mediated glomerular disorders isless studied thus far. Since the development of the AcP-IgAN model inmice involves an enhancement of inflammatory response locally in thekidney, whether NALP3 inflammation activation was operating in theAcP-IgAN mice was determined. At both day 3 and day 28, although greatlyincreased protein levels of NLRP3 was observed in the kidney of AcP-IgANmice, compared to normal controls, this effect was significantlyinhibited in AcP-IgAN+Antroq mice. mRNA expression levels of NLRP3 inboth control AcP-IgAN and AcP-IgAN+Antroq mice were greatly increased atday3, compared to normal controls, but Compound 1 administration showedno effects on the NLRP3 mRNA expression levels in the AcP-IgAN+Antroqmice. However, at day 28, AcP-IgAN+Antroq mice were found to havesubstantially reduced renal mRNA levels of NLRP3, compared to controlAcP-IgAN mice which still showed greatly higher renal mRNA levels of thegene, compared to normal controls. Importantly, renal mRNA expressionlevels of both caspase-1 and IL-18 was significantly elevated inAcP-IgAN+Antroq mice at as early as day 3 until later day 28, but thesetwo effects were greatly inhibited in AcP-IgAN+Antroq mice, asdemonstrated by quantitative real-time PCR analysis (FIGS. 16A-F). Inparallel, Antroq administration resulted in a greatly inhibited renalincrease in caspase-1 protein levels of AcP-IgAN+Antroq mice, comparedto those of control AcP-IgAN mice at both day 3 and day 28, althoughsignificantly inhibited renal IL-18 protein production inAcP-IgAN+Antroq mice was observed only at day 28. At day 28, controlAcP-IgAN mice revealed a greatly elevated mRNA levels of IL-1beta, butthis effect was significantly inhibited in AcP-IgAN+Antroq mice,although there was no detectable increase of renal IL-1beta mRNAexpression in all mice at day 3.

Renal NE-κB Activation and its Related Cytokines

Based on the prominent mononuclear leukocytic infiltration in the kidneyof AcP-IgAN mice, an active inflammatory response locally in the kidneyappeared to be an important pathway in response to the acceleration andprogression of IgAN. The role of NF-κB in the kidney was studies andpresented herein. First, as shown in FIGS. 17A-F, compared to controlAcP-IgAN mice (which showed significantly increased renal levels ofnuclear NF-kB protein at day 28 compared to normal controls),AcP-IgAN+Antroq mice exhibited significantly decreased levels of nuclearNF-kB protein in the kidney, although earlier at day 3, there was nodetectable increase in renal levels of the protein in both controlAcP-IgAN and AcP-IgAN+Antroq mice, compared to normal controls. Thiseffect of Compound 1 was further confirmed by a significantly reducedNF-kB nuclear translocation in AcP-IgAN+Antroq mice, compared to controlAcP-IgAN mice (FIGS. 17A and 17B), showing significantly increasednuclear translocation, as demonstrated by IHC of renal tissues at day28. Next, quantitative analysis of both renal mRNA and proteinexpression was performed for MCP-1 (FIG. 17C) and IL-6 (FIG. 17D),respectively. The Compound 1 administration significantly reduced therenal expression levels of mRNA and protein of MCP-1 and IL-6 inAcP-IgAN+Antroq mice, compared to control AcP+IgAN mice at day 28,although there was no such effect at day 3 between control AcP-IgAN,AcP-IgAN+Antroq and normal control mice (FIGS. 17E and 17F).

Apoptosis in the Kidney

Apoptosis in the kidney has been implicated in the pathogenesis of IgAN.As shown in FIGS. 18A-B, control AcP-IgAN mice showed significantlyincreased apoptosis in the kidney, as demonstrated by TUNEL, compared tonormal controls at day 28, but this effect was greatly inhibited byAntroq administration in AcP-IgAN+Antroq mice, although there was onlyinconspicuous apoptosis in all the mice examined early at day 3.

Example 15 Parenteral Formulation

To prepare a parenteral pharmaceutical composition suitable foradministration by injection, 100 mg of a compound or its salt describedherein is dissolved in DMSO and then mixed with 10 mL of 0.9% sterilesaline. The mixture is incorporated into a dosage unit form suitable foradministration by injection.

Example 16 Oral Formulation

To prepare a pharmaceutical composition for oral delivery, 100 mg of anexemplary Compound 1 was mixed with 100 mg of corn oil. The mixture wasincorporated into an oral dosage unit in a capsule, which is suitablefor oral administration.

In some instances, 100 mg of a compound described herein is mixed with750 mg of starch. The mixture is incorporated into an oral dosage unitfor, such as a hard gelatin capsule, which is suitable for oraladministration.

Example 17 Sublingual (Hard Lozenge) Formulation

To prepare a pharmaceutical composition for buccal delivery, such as ahard lozenge, mix 100 mg of a compound described herein, with 420 mg ofpowdered sugar mixed, with 1.6 mL of light corn syrup, 2.4 mL distilledwater, and 0.42 mL mint extract. The mixture is gently blended andpoured into a mold to form a lozenge suitable for buccal administration.

Example 18 Inhalation Composition

To prepare a pharmaceutical composition for inhalation delivery, 20 mgof a compound described herein is mixed with 50 mg of anhydrous citricacid and 100 mL of 0.9% sodium chloride solution. The mixture isincorporated into an inhalation delivery unit, such as a nebulizer,which is suitable for inhalation administration.

Example 19 Rectal Gel Formulation

To prepare a pharmaceutical composition for rectal delivery, 100 mg of acompound described herein is mixed with 2.5 g of methylcelluose (1500mPa), 100 mg of methylparapen, 5 g of glycerin and 100 mL of purifiedwater. The resulting gel mixture is then incorporated into rectaldelivery units, such as syringes, which are suitable for rectaladministration.

Example 20 Topical Gel Composition

To prepare a pharmaceutical topical gel composition, 100 mg of acompound described herein is mixed with 1.75 g of hydroxypropylcellulose, 10 mL of propylene glycol, 10 mL of isopropyl myristate and100 mL of purified alcohol USP. The resulting gel mixture is thenincorporated into containers, such as tubes, which are suitable fortopical administration.

Example 21 Ophthalmic Solution Composition

To prepare a pharmaceutical ophthalmic solution composition, 100 mg of acompound described herein is mixed with 0.9 g of NaCl in 100 mL ofpurified water and filtered using a 0.2 micron filter. The resultingisotonic solution is then incorporated into ophthalmic delivery units,such as eye drop containers, which are suitable for ophthalmicadministration.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is: 1-31. (canceled)
 32. A pharmaceutical compositionfor use in the treatment of glomerulosclerosis or glomerulonephritis ina subject comprising administering to the subject an effective amount ofa cyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur; R is ahydrogen or C(═O)C₁-C₈alkyl; each of R₁, R₂ and R₃ independently is ahydrogen, methyl or (CH₂)_(m)—CH₃; R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅,C(═O)R₅, halogen, 5 or 6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl,C₂-C₈alkynyl, aryl, glucosyl, wherein the 5 or 6-membered lactone,C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl areoptionally substituted with one or more substituents selected fromNR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈ alkoxy; eachof R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl; R₇ is aC₁-C₈alkyl, OR₅ or NR₅R₆; m=1-12; and n=1-12; or a pharmaceuticallyacceptable salt, metabolite, solvate or prodrug thereof.
 33. Apharmaceutical composition for use in attenuating renal dysfunction orglomerular lesions in a subject comprising administering to the subjectan effective amount of a cyclohexenone compound having the structure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur; R is ahydrogen or C(═O)C₁-C₈alkyl; each of R₁, R₂ and R₃ independently is ahydrogen, methyl or (CH₂)_(m)—CH₃; R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅,C(═O)R₅, halogen, 5 or 6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl,C₂-C₈alkynyl, aryl, glucosyl, wherein the 5 or 6-membered lactone,C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl areoptionally substituted with one or more substituents selected fromNR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈ alkoxy; eachof R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl; R₇ is aC₁-C₈alkyl, OR₅ or NR₅R₆; m=1-12; and n=1-12; or a pharmaceuticallyacceptable salt, metabolite, solvate or prodrug thereof.
 34. Apharmaceutical composition for use in (a) enhancing renal nuclear factorE2-related factor 2 (Nrf2) activity in a subject; (b) inhibiting renalNF-κB activation and/or transforming growth factor (TGF)-β1 proteinexpression in a subject; (c) inhibiting ROS/NO and/or p47^(phox) in asubject; (d) reducing CD3⁺/CD69⁺ T cells in a subject; (e) reducingpro-inflammatory cytokines in a subject; (f) maintaining immunoglobulinA nephropathy (IgAN) in remission in a subject; (g) enhancingglutathione peroxidase (GPx) activity in the kidney; (h) educing renalcaspase-1 protein expression and/or inhibiting renal NLRP3 activation inthe kidney; (i) reducing renal NF-κB level in the kidney; or (j)inhibiting apoptosis in the kidney; comprising administering to thesubject an effective amount of a cyclohexenone compound having thestructure:

wherein each of X and Y independently is oxygen, NR₅ or sulfur; R is ahydrogen or C(═O)C₁-C₈alkyl; each of R₁, R₂ and R₃ independently is ahydrogen, methyl or (CH₂)_(m)—CH₃; R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅,C(═O)R₅, halogen, 5 or 6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl,C₂-C₈alkynyl, aryl, glucosyl, wherein the 5 or 6-membered lactone,C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl areoptionally substituted with one or more substituents selected fromNR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈ alkoxy; eachof R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl; R₇ is aC₁-C₈alkyl, OR₅ or NR₅R₆; m=1-12; and n=1-12; or a pharmaceuticallyacceptable salt, metabolite, solvate or prodrug thereof.
 35. Thecomposition of claim 34, wherein the pro-inflammatory cytokines compriseMCP-1, IL-6, IL-1β, IL-18, or combinations thereof.
 36. A pharmaceuticalcomposition for use in protecting or preventing kidney fromglomerulosclerosis and/or glomerulonephritis in a subject comprisingadministering to the subject an effective amount of a cyclohexenonecompound having the structure

which decreases the expression levels of TGF-β1 protein and collagen I,III and IV protein accumulation in the kidney, wherein each of X and Yindependently is oxygen, NR₅ or sulfur; R is a hydrogen orC(═O)C₁-C₈alkyl; each of R₁, R₂ and R₃ independently is a hydrogen,methyl or (CH₂)_(m)—CH₃; R₄ is NR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅,halogen, 5 or 6-membered lactone, C₁-C₈alkyl, C₂-C₈alkenyl,C₂-C₈alkynyl, aryl, glucosyl, wherein the 5 or 6-membered lactone,C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, and glucosyl areoptionally substituted with one or more substituents selected fromNR₅R₆, OR₅, OC(═O)R₇, C(═O)OR₅, C(═O)R₅, C₁-C₈ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₈ cycloalkyl, C₁-C₈ haloalkyl, and C₁-C₈ alkoxy; eachof R₅ and R₆ is independently a hydrogen or C₁-C₈alkyl; R₇ is aC₁-C₈alkyl, OR₅ or NR₅R₆; m=1-12; and n=1-12; or a pharmaceuticallyacceptable salt, metabolite, solvate or prodrug thereof.
 37. Thepharmaceutical composition of claim 32 for use in the treatment ofglomerulosclerosis that is focal segmental glomerulosclerosis (FSGS)wherein said compound (i) enhances Nrf2 activity and/or (ii) suppressesNF-κB-dependent inflammatory and TGF-β1-mediated fibrosis in the kidney.38. The pharmaceutical composition of claim 32 for use in the treatmentof glomerulonephritis wherein said compound (i) blocking renal NLRP3inflammasome activation and/or (ii) inhibiting the increase in T cellactivation in the subject.
 39. The composition of claim 32, whereinglomerulosclerosis is focal segmental glomerulosclerosis (FSGS) ornodular glomerulosclerosis.
 40. The composition of claim 32, whereinglomerulosclerosis is focal segmental glomerulosclerosis (FSGS).
 41. Thecomposition of claim 32, wherein glomerulonephritis is immunoglobulin Anephropathy (IgAN).
 42. The composition of claim 32, wherein saidcyclohexenone compound blocks oxidative stress.
 43. The composition ofclaim 42, wherein the oxidative stress is blocked by reducing TGF-β1 andextracellular matrix protein expression.
 44. The composition of claim32, wherein the cyclohexenone compound reduces CD3⁺/CD69⁺ T cells orpro-inflammatory cytokines in the subject.
 45. The composition of claim44, wherein the pro-inflammatory cytokines comprise MCP-1, IL-6, IL-1β,IL-18, or combinations thereof.
 46. The composition of claim 33, whereinsaid glomerular lesions comprise epithelial hyperplasia lesion (EPHL).47. The composition of claim 32, wherein said compound is isolated fromAntrodia camphorate.